農桿菌介導的葡萄胚性與非胚性愈傷組織遺傳轉化過程中脅迫響應基因的表達

摘 要： 葡萄愈傷組織經農桿菌侵染后會產生細胞褐化和壞死現象，大大降低了轉基因效率，此過程中脅迫基因發揮了重要作用。我們通過sqRT-PCR的方法研究了葡萄胚性（EC）和非胚性（NEC）愈傷組織在農桿菌侵染前、侵染30 min以及共培養3 d后PR-10， Mn-SOD， APX， IRL 5， CAT和β-1，3-glucanases基因的表達情況。EC和NEC中PR-10表達量均較高，并在侵染過程中呈上升趨勢；NEC的Mn-SOD表達量沒有明顯變化，EC侵染30 min后Mn-SOD表達量的相對灰度值提高為侵染前的1.8倍，共培養3 d后又降低為侵染前的0.9倍；NEC的APX表達量低且比較穩定，EC在侵染前、侵染30 min和共培養3 d后APX表達量的相對灰度值分別為NEC的3.2倍、2.1倍和4.1倍；NEC中CAT表達量較低，且在轉基因過程中呈現逐漸降低的趨勢，EC在侵染30 min后CAT表達量的相對灰度值提高為侵染前的2.4倍；NEC中IRL 5的表達量非常低，和CAT一樣在轉基因過程中呈現下降趨勢，而EC中該基因的表達量相對高一些；NEC的β-1，3-glucanases表達量遠遠高于EC，侵染前、侵染30 min和共培養3 d后其表達量的相對灰度值分別為EC的29倍、5.5倍和4.7倍。我們還研究了蛋白酶處理以后，這些基因在EC中的表達情況。結果顯示：共培養3 d后進行蛋白酶處理的EC中Mn-SOD，APX，IRL 5這3個基因表達量相對灰度值降低為未處理EC的0.21、0.74和0.10，CAT和β-1，3-glucanases的表達量分別提高為未處理EC的3.7倍和8.7倍。這些結果在一定程度上說明了葡萄胚性和非胚性愈傷對于農桿菌侵染呈現不同褐化反應的潛在原因，對于我們在基因水平上研究這2種愈傷組織經過農桿菌侵染后的細胞可塑性提供了新的視角，有利于將來進一步降低愈傷褐化和細胞死亡的研究，從而提高轉基因效率。

關鍵詞： 葡萄； 農桿菌； 轉化；sqRT-PCR； 脅迫反應；蛋白酶

中圖分類號：Ｓ６６3．1 文獻標識碼：Ａ 文章編號：１００９－９９８０?穴２０11?雪０6－964－０8

Expression of stress response genes in EC and NEC of Vitis vinifera during Agrobacterium-mediated transformation

ZHAO Feng-xia1，3，WANG Zheng-ping1，GAO Xiang-bin1，CHEN Shang-wu2， MA Hui-qin3

（1Tobacco Research Center，Henan Academy of Agricultural Sciences，Xuchang，Henan 461000 China； 2College of Food Science and Nutritional Engineering， China Agricultural University， Beijing 100083 China； 3College of Agriculture and Biotechnology， China Agricultural University， Beijing 100193 China）

Abstract: Callus of grape inoculated with Agrobacterium tumefaciens often suffer cell browning and necrosis， which greatly reduces the efficiency of transformation. Stress response genes seem to play key roles during this process. Using semi-quantitative reverse transcription-polymerase chain reaction （sqRT-PCR）， we studied the expression levels of pathogenesis-related protein 10 （PR-10）， manganese-superoxide dismutase （Mn-SOD）， ascorbate peroxidase （APX）， isoflavone reductase-like protein 5 （IRL 5）， catalase （CAT） and β-1， 3-glucanases genes in embryogenic callus （EC） and non-embryogenic callus （NEC） of Vitis vinifera L. at 0 min， 30 min and 3 d after inoculation with A. tumefaciens. The expression level of PR-10 gene was quite high in both NEC and EC and gradually increased during co-cultivation. Mn-SOD in NEC had no obvious change， while the relative expression level of Mn-SOD in EC 30 min after inoculation increased to 1.8 times that of EC before inoculation. At 3 d of co-cultivation， it reduced to 0.9 times. The expression of APX in NEC was stable and low during the process， while the expression level in EC before inoculation， 30 min and 3 d after inoculation was 3.2， 2.1 and 4.1 times higher compared to the corresponding level in NEC， respectively. The expression of CAT in NEC was very low and gradually declined. The expression in EC 30 min after inoculation increased by 2.4 times compared to that before inoculation. IRL 5 presented the same decreasing tendency in NEC as CAT， and its expression was relatively high in EC. The expression of β-1，3-glucanases was much higher in NEC than EC. Before inoculation， 30 min after inoculation and 3 d after co-cultivation， the relative expression intensity of β-1，3-glucanases in NEC was 28， 4.5 and 3.7 times higher than in EC. Further more， the expression of these genes in EC treated with protease was also detected. After 3 d of co-cultivation， the expression of Mn-SOD， APX and IRL 5 in protease treated EC were reduced， but the expression of CAT and β-1，3-glucanases increased to 3.7 and 8.7 times as high as that in EC without protease treatment， respectively. The results， to a certain extent， unraveled the mechanism underlying the difference in reactions to A. tumefaciens between EC and NEC and provided a new insight into Agrobacterium-induced cell plasticity at gene level， which could be useful for strategies to reduce callus browning and cell death and thus improve grape transformation efficiency.

Key words: Vitis vinifera L.； Agrobacterium tumefaciens；Transformation； sqRT-PCR； Stress response；Protease

Grape is highly heterozygous and conventional breeding processes are very time- and labor-consuming[1]. As a result， genetic transformation is highly demanded by grape industry. Since the first transgenic grape （Vitis vinifera L.） was reported two decades ago， significant achievements have been obtained in grape transformation for disease resistance[2] and fruit quality improvement. Agrobacterium-mediated transformation is a widely used method for grapevine genetic engineering. EC induced from anther are commonly used materials during transformation[3]. Many researchers studied the regeneration system and the Agrobacterium-mediated transformation of grapevine using EC as initiatory materials. However， NEC， which lacks the ability of embryogenesis， is inevitably formed and develops during the induction and subculture of EC[4]. It is different from EC anatomically， physiologically， biochemically and epigenetically. NEC proliferates fast， but also keeps in a rather stable condition. The transformation of NEC would help to understand the key genes or transcription factors that distinguish EC and NEC， validating the differentially expressed genes found in previous molecular biological studies[5].

EC exposed to A. tumefaciens often suffer browning and necrosis， which drastically reduces the transformation efficiency. Interestingly， this hypersensitive-like necrotic response appears to be less serious in NEC， which suggests that certain physiological conditions underlying embryogenic potential in EC may be related to type of response to A. tumefaciens inoculation. Understanding the regulatory mechanisms of the Agrobacterium-induced necrotic reaction in grapevine is urgently required.

This paper focused on the expression levels of stress response genes during Agrobacterium-mediated transformation in EC and NEC of grape cv. Cabernet Sauvignon in order to understand their stress responses to A. tumefaciens.

1 Materials and Methods

1.1 The preparation of plant materials and A. tumefaciens mediated- transformation

EC and NEC of V. vinifera L. were obtained according to previous study[6]. In brief， anther from flower buds 12 to 14 d before anthesis were collected， sterilized and placed on an anther inoculation medium. When EC and NEC became visually separated， subcultured them on solid maintenance medium.

A vector pA5 containing a chitinase signal sequence fused to gfp and a HDEL motive[7] was introduced into A. tumefaciens strain EHA 105. A share of 5 g callus was immersed into A. tumefaciens （D600 nm=1.0） containing the vector pA5， cultured for 30 min and then transferred onto a piece of filter paper on a co-cultivation medium. After 3 d of co-cultivation at 25 ℃ in darkness， the callus was collected from the filter paper， washed three times with liquid maintenance medium supplemented with 1 g·L-1 cefotaxime and 100 mg ·L-1 dithiothreitol， and transferred onto a solid maintenance medium.

1.2 Quantitative analyses of transformation efficiency

Expression of the gfp gene in EC and NEC was observed from 10 d after A. tumefaciens infection， under a laser confocal scanning microscope （Nikon D-ECLIPSE C1; Nikon， Tokyo， Japan）. Transformation efficiency was calculated as the number of GFP spots （individual cells or multicellular aggregates） per gram of callus according to Lopez-Perez[8].

1.3 Sequence characterization and primer design of representative stress response genes

PR-10 （GI: 163914212）， Mn-SOD （GI:161778781）， APX （GI:73647737）， IRL 5 （GI:76559893）， CAT （GI:19070129）， β-1，3-glucanases （GI:6273715） and ACT1 （GI:149938963） genes sequence were retrieved from the databases of the Genoscope （http://genoscope.cns.fr/vitis）， IASMA （http://genomics.research.iasma.it/iasma）， NCBI and Genbank according to their counterparts from Arabidopsis thaliana and other crops. Primers were designed according to the conservative sequences.

1.4 sqRT-PCR for the expression analysis of stress response genes

For RNA extraction， samples were taken from the following three stages: EC and NEC before inoculation （EC0， NEC0）， 30 min after inoculation with A. tumefaciens （EC1， NEC1）[6]， and 3 d of co-cultivation （EC2， NEC2）. In addition， the expression of the six genes in EC treated with 20 mg·L-1 protease in liquid and solid co-cultivation media during the 30 min inoculation and the following 3 d co-cultivation were also detected. They were sampled after 3 d of co-cultivation and marked as EC （Pro）. All the infection treatments were carried out with three replications. Samples were transferred in liquid nitrogen and further conserved at -80 ℃ for RNA extraction.

Total RNA was extracted from all the samples with Trizol （Invitrogen， USA）. The resulting RNA samples were treated with RNase-free DNase I to avoid the potential genomic DNA contamination. For the sqRT-PCR， cDNA strands were synthesized using the Superscript First-Strand Synthesis System for RT-PCR （Invitrogen）. Primers were designed based on the mRNA or EST sequences in the stress response genes. Control reactions to normalize sqRT-PCR amplification were run with ACT1 （Accession: EF063572.1） specific primers （Table 1）.

All sqRT-PCR amplifications were performed in triplicate for each RNA sample. The amplified fragments were detected by UV after electrophoresis on agarose gel （1% w/v） with goldview （Peiqing Technology， Shanghai， China） and gene expression levels were quantified as band fluorescence intensity in agarose gels after electrophoresis.

2 Results and Analysis

2.1 The establishment and verification of EC and NEC

Grape anther explants yielded two types of callus tissue on callus induction medium; the characters of EC and NEC were in agreement with previous reports. EC had a soft and friable tissue structure and was light yellow in color （Plate-A）， while NEC was characterized by a loose and watery structure， with translucent and shiny appearance （Plate-B）. On the solid maintenance medium， EC and NEC kept their specific characters. The proliferation rate of the NEC line was significantly higher than that of the EC line. When the two cell lines were inoculated on regeneration medium respectively， EC developed into small somatic embryos after about 30 d of culture （Plate-C）， while NEC maintained the uniform and un-organized appearance （Plate-D ）.

2.2 The observation of GFP fluorescence in transformed cells

Transformed EC and NEC were observed under a laser confocal scanning microscope after 2 rounds of subculture on selection medium. Bright green fluorescence was observed and uniform GFP expression in cells was presented separately （Plate-E~F）. EC-GFP cells were smaller， marked with clusters of small vacuoles， and organelles located near the nucleus （Plate-E）. NEC-GFP cells were oval or rhabditiform in shape， with the nucleus located in a narrow strip of cytoplasm along the cell wall and a large centralized vacuole （Plate-F）.

2.3 The expression of stress response genes with normal transformation protocol

We conducted a comprehensive expression analysis of V. vinifera L. Cabernet Sauvignon EC and NEC with focus on stress response genes， preliminarily to unveil their mechanisms underlying their differential responses to Agrobacterium-mediated transformation. The transcription levels of these six representative genes were analyzed by sqRT-PCR （Fig.1~2）. Differential expression of the genes， PR-10， APX， IRL 5， Mn-SOD， β-1，3-glucanases and CAT in the EC and NEC cells indicated their differential responses to A. tumefaciens.

In our research， the expression of PR-10 gene was high in the two types of callus but much higher in NEC than in EC （Fig.1~2）. During inoculation， the expression of PR-10 gene was greatly increased. As a result， they presented a gradual increasing trend in both EC and NEC.

Plant cells were protected against the damage from reactive oxygen species （ROS） generated during the hypersensitive response by a complex antioxidant system including actions of antioxidant enzymes such as SOD， POX， and CAT[9]. In our study， the expression of Mn-SOD in both EC and NEC increased initially and then decreased. It had no obvious change in NEC before and after transformation （Fig.2）， which was different from EC （Fig.1）. The expression of Mn-SOD in EC0 and EC2 was low but very high in EC1. After co-cultivation for 3 d， ROS homeostasis in these callus was likely reached. As a result， the expression of Mn-SOD decreased.

APX gene was one of the defense genes which are closely related to disease resistance. It plays an important role in defense reactions and is also a significant component in AsA-GSH cycle. In the publication of grape somatic embryo （SE） proteome， two APX spots with an acidic isoelectric point were demonstrated， which were two times heavier in EC than in NEC[10]. In our study， the expression of APX in EC0 was more than 3 times higher than in NEC0 （Fig.1~2）. It was in agreement with Marsoni’s proteome result in grape. During transformation， APX in NEC was weakly expressed （Fig. 2）， while for EC， its expression was much higher （Fig. 1）. The expression of APX in EC was quite high before inoculation and it decreased a little after 30 min inoculation. Its expression elevated again and reached the highest after 3 d of co-cultivation.

In a previous study， the content of CAT protein was found more than 2 times higher in NEC than in EC[10]， and similar results were obtained in this study （Fig.1~2）. The expression of CAT in NEC presented a gradual decreasing tendency during transformation. NEC without inoculation had the highest expression. CAT expression reduced a little bit after 30 min of inoculation and became the lowest at 3 d of co-cultivation. The change pattern in EC was quite different from that in NEC. The expression in EC without inoculation was the lowest， while it rapidly increased after 30 min inoculation. In EC2， it was slightly lower than in EC1 but much higher than in EC0.

In our study， the expression of IRL 5 gene in NEC was very low and gradually declined （Fig. 2）. But its expression was comparatively high in EC （Fig. 1）.

The expression of β-1，3-glucanases gene was much higher in NEC0 than EC0 （Fig.1~2）. And during transformation， it increased greatly in NEC. After 3 d of co-cultivation， the expression was increased by over 3 folds. It was much lower in EC， although it had a tiny increase after inoculation. High level of β-1，3-glucanases might inhibit bacteria from entering the callus， which contributes to disease resistance.

2.4 The expression of stress response genes during transformation with protease

The use of protease at 20 mg·L-1 in the liquid and solid co-cultivation media for the 30 min of inoculation and the following 3 d of co-cultivation respectively seriously reduced the necrosis of callus and significantly improved the transformation efficiency in many plants （data not shown）. The expression of stress response genes had significant differences between EC with and without protease treatment during transformation （Fig. 3）.

The expression of Mn-SOD， APX and IRL 5 genes reduced after co-cultivation with protease. Expression of Mn-SOD and IRL 5 became hardly detectable. The expression of APX in EC（Pro） was much lower than in EC2. PR-10 was greatly increased in EC co-cultivated with protease than EC before infection. It was notable that the expression of CAT and β-1，3-glucanases presented an astonishing increase. CAT expression intensity was nearly 4 times higher in EC（Pro） than in EC2 and β-1，3-glucanases expression intensity almost 9 times higher.

3 Discussions

Exposure of grape EC and NEC to A. tumefaciens leads to cell browning and necrosis. Plant tissue necrosis and cell death has been reported to be one of the major factors that severely reduce the efficiency of Agrobacterium-mediated transformation in many crops[4]. As a consequence， the genetic transformation is still a challenge. Studies have shown that EC is more susceptible to this hypersensitive-like necrotic response than NEC[4].

Plant defense mechanisms are complex and multiple factors are involved in stress resistance. ROS are generated soon after plant–pathogen contact resulting in the so-called oxidative burst[11]. Oxidative burst or ROS generation is often the first detectable response to biotic or environmental stresses in plants[12]. The burst of ROS is indispensable in the reprogramming of cell metabolism as an adaptation response to stresses， influencing the expression of stress-associated genes. The molecular response of plants to stresses has often been considered a complex process based mainly on the modulation of transcriptional activity of stress-related genes.

PR-10 was a relatively new member of PR genes and has been characterized in recent years[13]. Enhanced PR-10 expression after pathogen attack had been found in rice[14]， sorghum[15]， and V. vinifera L.[16]. It appears to be primarily induced by ROS. Previous studies with an asparagus PR-10 gene （AoPR10） promoter in heterologous plants suggested that the AoPR10-GUS transgene was responsive to oxidative signals/stresses[17]. ROS also induce the expression of endogenous PR-10 genes from Arabidopsis[17]. They are often expressed in response to abiotic and biotic stresses and may have RNase activities that could play an antiviral role[18].

Apart from pathogen-inducible expression， PR-10 gene is also expressed in an organ- or tissue-specific manner during development in healthy plants[19]. Some researchers suggested that PR-10 protein was involved in plant growth and development[18]. They may take part in the steroid hormone-mediated disease resistance in plant defense response.

In a previous study of our group， PR-10 protein was found to be the most abundant protein in both EC and NEC， with astonishingly high levels in NEC[4]. In this research， the expression of PR-10 gene was high in both types of callus and was much higher in NEC than in EC. The result was in good agreement with the relative protein volume in the 2D-gels of the EC and NEC samples[4]. The expression of PR-10 gene was activated by pathogen infection as part of plant defense response. The higher expression of PR-10 gene in NEC than in EC was one of the reasons that NEC was more resistant to pathogen and less susceptible to necrosis.

Plant cells are protected against damage from ROS generated during the hypersensitive response by a complex antioxidant system[20]. SOD， APX and CAT work as key players in the modulation of ROS levels and ROS mediated stress signaling. The function of SOD is to convert to H2O2. Oxidative burst is thought to be induced by the accumulation of SOD， which resulted in stress response[21].

As an adaptive response to stress， the increased expression of Mn-SOD immediately after inoculation may play a key role in the modulation of ROS levels and ROS mediated stress signaling. The rapid rise of Mn-SOD in EC after 30 min of inoculation increased the concentration of H2O2. Low concentration of H2O2 induces the expression of defense genes[22]. It might have activated the defense reaction in the callus exposed to A. tumefaciens. While high concentration of H2O2 induces cell death. This was one of the reasons why EC cells were more susceptible to necrosis.

H2O2 was degraded by CAT and APX. Recent results from studies in Nicotiana demonstrated that CAT activity decrease was correlated with pathogen stress-related ROS generation and hypersensitive cell death， while the expression of CAT was correlated with increased tolerance to ROS[23]. CAT decomposes H2O2 into H2O and O2 in peroxisomes and plays an important role in stress-induced cell apoptosis. Immediately after 30 min of inoculation， the expression of CAT in EC increased， as a result， the ability of EC in clearing away and generating H2O2 was somewhat greater than NEC. The concentration of H2O2 may be higher in EC， which would induce more serious cell death. After 3 d of co-cultivation， the expression of SOD and CAT in EC was greatly decreased， while APX increased. produced during the process had not been cleared away in time thus might be accumulated. In the same time， H2O2 was degraded. is the main form of ROS and it has the ability of killing the bacteria and cells themselves. The accumulation of also induces the expression of APX[24]. It explained from another aspect why EC is more susceptible to browning and necrosis than NEC during the Agrobacterium-mediated transformation. In NEC， the expression of Mn-SOD， APX and CAT were stable. The production and elimination of ROS was relatively balanced and NEC was less necrotic.

Isoflavonoid phytoalexins are associated with plant defense. Isoflavone reductase （IFR） specifically recognized isoflavones. IFRs are restricted primarily to legumes and IRL proteins have significant sequence homology to legume IFRs. IRL proteins may be enzymes catalyzing distinct reduction reactions， and their expression can be induced by oxidative stress[25]. In a previous study， three spots were identified as IRL 5 and all of them were only detectable in EC samples[4]. In our study， the expression of IRL 5 gene in NEC was very low while it was comparatively high in EC. It was consistent with the result of the previous study in our group.

A wide variety of enzymes have been found associated with disease resistance， and hydrolytic enzymes may have a dual function in this process: some isozymes have direct antimicrobial effects against an invading pathogen. These isozymes may also accelerate and amplify the disease resistance process by generating hypersensitive response elicitors upon encountering a pathogen. Soybean β-1，3-glucanases[26] and specific isozymes of tomato chitinase and β-1，3-glucanase[27] have been demonstrated to generate elicitors from fungal pathogens. Interestingly， the β-1，3-glucanases accumulated in cultivars of resistant wheat could be involved in resistance to the Russian wheat aphid[28]. Glucanases may also govern plant developmental processes which are not directly related to pathogenesis or stress resistance. In our study， the expression of β-1，3-glucanases was greatly enhanced both in grape EC and NEC. Increases in the expression and activity of β-1，3-glucanases after induction had also been reported in rice[29]. Accumulation of β-1，3-glucanases was observed in infected spruce seedlings. Similarly， immunized plants produced β-1，3-glucanases and other pathogenesis-related proteins in greater quantities than nonimmunized plants[30]. The expression of β-1，3-glucanases gene in NEC was higher than that in EC. Our result proved that NEC had greater resistance to A. tumefaciens than EC and thus they did not exhibit serious browning and necrotic reaction during co-cultivation with A. tumefaciens.

Previous study demonstrated that necrosis was mainly induced by a 32 ku harpin-like heat-stable protein （data not shown）. Transient GUS expression was greatly improved in banana， wheat， tomato and grape following co-cultivation in the presence of protease type XIV from Streptomyces griseus and they did not induce necrosis. Co-cultivation with protease significantly decreased the expression of Mn-SOD， APX， IRL 5 and increased the expression of CAT and β-1，3-glucanases in EC（Pro）. The expression of CAT was related with increased tolerance to ROS and the expression of β-1，3-glucanases inhibited bacteria from entering the calli. Therefore， inoculation and co-cultivation with protease could reduce necrosis and improve the transformation efficiency.

4 Conclusion

Using sqRT-PCR， we detected the expression levels of six key stress-response genes in EC and NEC subjected to normal transformation protocol， and EC co-cultivated with protease. The results unraveled the mechanism behind the different responses of the two types of callus to A. tumefaciens inoculation. They also explained why EC infected with protease treatment reduced necrosis and cell death. （本文圖版見插5）

References:

[1] BORNHOFF B A， HARST M， ZYPRIAN E， TOPFER R. Transgenic plants of Vitis vinifera cv. Seyval blanc[J]. Plant Cell Reports， 2005， 7: 433-438.

[2] HERRERAESTRELLA L， SIMPSON J. Genetically-engineered resistance to bacterial and fungal pathogens[J]. World Journal of Microbiology Biotechnology， 1995， 4: 383-392.

[3] PERL A， SAAD S， SAHAR N， HOLLAND D. Establishment of long-term embryogenic cultures of seedless Vitis-vinifera cultivars - a synergistic effect of auxins and the role of abscisic-acid[J]. Plant Science， 1995， 2: 193-200.

[4] ZHANG J W，MA H Q，CHEN S，JI M，PERL A，KOVACS L，CHEN S W. Stress response proteins’ differential expression in embryogenic and non-embryogenic callus of Vitis vinifera L. cv. Cabernet Sauvignon-A proteomic approach[J]. Plant Science，2009，2: 103-113.

[5] CHEN S， ZENG L， CHEN S W， SUN Y W， ZHANG W， XU H Y， MA H Q. Differentiated expression of VvSUC12 and VvSUC27 in embryogenic and non-embryogenic calli of Vitis vinifera L.[J]. Chinese Journal of Biotechnology/Shengwu Gongcheng Xuebao， 2010， 4: 530-537.

[6] ZHAO F A， CHEN S W， PERL A， DAI R， XU H Y ， MA H Q. The establishment of an Agrobacterium-mediated transformation platform for the non-embryogenic calli of Vitis vinifera L.[J]. Agricultural Sciences in China， 2011， 5: 101-105.

[7] SIEMERING K R， GOLBIK R， SEVER R， HASELOFF J. Mutations that suppress the thermosensitivity of green fluorescent protein[J]. Current Biology， 1996， 12: 1653-1663.

[8] LOPEZ-PEREZ A J，VELASCO L，PAZOS-NAVARRO M， DABAUZA M. Development of highly efficient genetic transformation protocols for table grape sugraone and grimson seedless at low agrobacterium density[J]. Plant Cell Tissue and Organ Culture， 2008， 2: 189-199.

[9] ZHANG J X， KIRKHAM M B. Drought-stress-induced changes in activities of superoxide-dismutase， catalase， and peroxidase in wheat species[J]. Plant and Cell Physiology， 1994， 5: 785-791.

[10] MARSONI M， BRACALE M， ESPEN L， PRINSI B， NEGRI A S， VANNINI C. Proteomic analysis of somatic embryogenesis in Vitis vinifera[J]. Plant Cell Reports， 2008，27: 347-356.

[11] LAMB C， DIXON R A. The oxidative burst in plant disease resistance[M]. Annual Review of Plant Physiology and Plant Molecular Biology， 1997: 251-275.

[12] PRAPAGDEE B， VATTANAVIBOON P， MONGKOLSUK S. The role of a bifunctional catalase-peroxidase KatA in protection of Agrobacterium tumefaciens from menadione toxicity[J]. Fems Microbiology Letters， 2004， 2: 217-223.

[13] LEE S C， HWANG B K. Induction of some defense-related genes and oxidative burst is required for the establishment of systemic acquired resistance in Capsicum annuum[J]. Planta，2005， 6: 790-800.

[14] MCGHEE J D， HAMER J E， HODGES T K. Characterization of a PR-10 pathogenesis-related gene family induced in rice during infection with Magnaporthe grisea[J]. Molecular Plant-Microbe Interactions， 2001， 7: 877-886.

[15] FLORES H E， VIVANCO J M， LOYOLA-VARGAS V M. Radicle biochemistry: the biology of root-specific metabolism[J]. Trends in Plant Science， 1999， 6: 220-226.

[16] BONOMELLI A， MERCIER L， FRANCHEL J， BAILLIEUL F， BENIZRI E， MAURO M C. Response of grapevine defenses to UV-C exposure[J]. Am J Enol Vitic， 2004， 55: 51-59.

[17] MUR L A J， STURGESS F J， FARRELL G G， DRAPER J. The AoPR10 promoter and certain endogenous PR10 genes respond to oxidative signals in Arabidopsis[J]. Molecular Plant Pathology， 2004， 5: 435-451.

[18] KIM S T， YU S， KANG Y H， KIM S G， KIM J Y， KIM S H， KANG K Y. The rice pathogen-related protein 10 （JIOsPR10） is induced by abiotic and biotic stresses and exhibits ribonuclease activity[J]. Plant Cell Reports，2008，3: 593-603.

[19] LIU J J， EKRAMODDOULLAH A K M， YU X S. Differential expression of multiple PR10 proteins in western white pine following wounding， fungal infection and cold-hardening[J]. Physiologia Plantarum， 2003， 4: 544-553.

[20] KUTA D D， TRIPATHI L. Agrobacterium-induced hypersensitive necrotic reaction in plant cells: a resistance response against Agrobacterium-mediated DNA transfer[J]. African Journal of Biotechnology， 2005， 8: 752-757.

[21] DENLIN W S， GUSTINE D L. Involvement of the oxidative burst in phytoalexin accumulation and the hypersensitive reaction[J]. Plant Physiol， 1992， 100: 1189-1195.

[22] WANG C， SHI F M. Relationship between ROS and NO under adversity stress[J]. Journal of Anhui Agricultural Science，2009，5: 1903 -1904.

[23] BARNA B， SMIGOCKI A C， BAKER J C. Transgenic production of cytokinin suppresses bacterially induced hypersensitive response symptoms and increases antioxidative enzyme levels in Nicotiana spp. [J]. Phytopathology， 2008， 11: 1242-1247.

[24] JIANG M Y， ZHANG J H. Water stress-induced abscisic acid accumulation triggers the increased generation of reactive oxygen species and up-regulates the activities of antioxidant enzymes in maizeleaves[J]. Journal of Experimental Botany，2002，53: 2401-2410.

[25] BABIYCHUK E， KUSHNIR S， BELLESBOIX E， VANMONTAGU M， INZE D. Arabidopsis-thaliana nadph oxidoreductase homologs confer tolerance of yeasts toward the thiol-oxidizing drug diamide[J]. Journal of Biological Chemistry， 1995， 44: 26224-26231.

[26] HAM K S， KAUFFMANN S， ALBERSHEIM P， DARVILL A G. Host-pathogen interactions.39. A soybean pathogenesis-related protein with Beta-1，3-Glucanase activity releases phytoalexin elicitor-active heat-stable fragments from fungal walls[J]. Molecular Plant-Microbe Interactions， 1991， 6: 545-552.

[27] LAWRENCE C B， SINGH N P， QIU J S， GARDNER R G， TUZUN S. Constitutive hydrolytic enzymes are associated with polygenic resistance of tomato to Alternaria solani and may function as an elicitor release mechanism[J]. Physiological and Molecular Plant Pathology， 2000， 5: 211-220.

[28] LINTLE M， VAN DER WESTHUIZEN A J. Glycoproteins from russian wheat aphid infested wheat induce defence responses[J]. Zeitschrift Fur Naturforschung C-a Journal of Biosciences， 2002（9/10）: 867-873.

[29] MANANDHAR H K， MATHUR S B， SMEDEGAARD-PETERSEN V， THORDAL-CHRISTENSEN H. Accumulation of transcripts for pathogenesis-related proteins and peroxidase in rice plants triggered by Pyricularia oryzae， Bipolaris sorokiniana and u.v. light[J]. Physiological and Molecular Plant Pathology，1999，5: 289-295.

[30] LAWRENCE C B， JOOSTEN M H A J， TUZUN S. Differential induction of pathogenesis-related proteins in tomato by Alternaria solani and the association of a basic chitinase isozyme with resistance[J]. Physiological and Molecular Plant Pathology，1996， 6: 361-377.