摘要:通過農桿菌介導轉入擬南芥(Arabidopsis thaliana)中進行過量表達。PCR及Western-blot分析結果表明,ZmLTP3基因已轉入擬南芥中,并且在大多數轉化植株中穩定表達。
關鍵詞:玉米(Zea mays);ZmLTP3基因;擬南芥(Arabidopsis thaliana);遺傳轉化
中圖分類號:Q782 文獻標識碼:A 文章編號:0439-8114(2014)04-0929-03
Construction and Transformation of Maize ZmLTP3 Gene Expression Vector
SUN Xiao-yan,ZHU Yong,ZOU Hua-wen
(College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China)
Abstract: LTP3 plays important roles in plant physiology. To study the function of a LTP3-like maize(Zea mays) ZmLTP3 gene, the cDNA of ZmLTP3 was constructed into the expression vector pGreen0029 and over-expressed in Arabidopsis thaliana. PCR and Western-blot analysis showed that ZmLTP3 gene was transformed into A. thaliana and expressed in majority transgenic plants.
Key words: maize(Zea mays); ZmLTP3 gene; Arabidopsis thaliana; transformation
植物轉脂蛋白(Lipid transfer proteins,LTPs)是一類小分子量、多基因家族編碼的的堿性蛋白質,具備轉移磷脂和脂肪酸的能力[1-3]。轉脂蛋白分為兩個亞家族,即LTP1和LTP2。LTP1亞家族成員含有90~95個氨基酸殘基,分子質量為9 ku左右;LTP2亞家族成員通常含有70個氨基酸殘基,分子質量為7 ku左右[4]。轉脂蛋白家族成員的結構高度保守:分子內部含有兩個保守的五肽基序(Thr/Ser-X1-X2-Asp-Arg/Lys、Pro-Tyr-X-Ile-Ser)。另外,蛋白質分子中都含有8個保守的半胱氨酸,形成4對二硫鍵[5];轉脂蛋白分子還含有一個由4個α-螺旋形成的疏水洞穴,可以和所轉運的磷脂或脂肪酸相互作用,從而行使其生物學功能[6]。
目前為止,已經從多種植物中被克隆、鑒定了轉脂蛋白基因[7-12]。除了擁有最初認為的脂轉移功能外,還發現具有其他多種生物學功能,如種子脂肪動員、體細胞發育、蠟物質形成、表皮形成、花粉管黏著、抗病等[4,13-19]。相對于在生物脅迫中的功能而言,轉脂蛋白在非生物脅迫中功能的直接證據還鮮有報道。
在此前的研究中,課題組相繼從玉米(Zea mays)中克隆了Pto/Pti1信號傳導途徑的兩個重要成員ZmPto和ZmPti1基因,過量表達ZmPto和ZmPti1基因的擬南芥(Arabidopsis thaliana)株系表現出較高的抗鹽性[20,21]。隨后,對轉基因擬南芥株系做基因芯片分析。結果表明,轉基因擬南芥中LTP3基因(At5g59320)的表達量相對于野生型提高了近18倍。這表明LTP3可能是Pto/Pti1信號系統的下游組分,并且在抗鹽過程中起到非常重要的作用。隨后,從玉米中克隆了擬南芥LTP3基因的同源基因ZmLTP3(GenBank Accession No. JX435819),RT-PCR分析發現ZmLTP3可由多種生物和非生物脅迫因子(尤其是高鹽)所誘導(待發表)。本研究構建了植物表達載體,將ZmLTP3基因轉入擬南芥中過量表達,為研究其生物學功能提供可能。
1 材料與方法
1.1 試驗材料
1.1.1 材料 擬南芥(Columbia 生態型)、大腸桿菌(E. coli) DH5α、農桿菌GV3101、pGEM-T載體、含ZmPti1-dHA序列的酵母表達載體p426GAL1、含35S-C4DDPK-CBF3-NOS片段的植物表達載體pGreen0029均由長江大學農學院保存。
1.1.2 試劑 DNA 凝膠回收試劑盒購自杭州V-gene生物技術公司; DNA限制性內切酶、T4 DNA 連接酶、ExTaq酶、DNA Marker均購自寶生物工程(大連)有限公司;Anti-HA(3f10)及Peroxidase-conjugated goat anti-rat IgG(H+L)均購自Roche公司;PVDF膜購自Millipore公司;其他試劑均為國產分析純。PCR 引物及DNA測序工作由生工生物工程(上海)股份有限公司完成。
1.2 試驗方法
1.2.1 ZmLTP3基因植物表達載體的構建 設計PCR引物(含BamHⅠ和StuⅠ酶切位點)擴增pGEM-T載體中ZmLTP3基因序列,BamHⅠ/StuⅠ雙酶切PCR產物和含ZmPti1-dHA序列的酵母表達載體p426GAL1。T4 DNA連接酶連接酶切后的載體與PCR產物,形成重組載體p426GAL1。隨后用BamHⅠ/PstⅠ雙酶切重組載體p426GAL1和含有35S-C4DDPK-CBF3-NOS片段的植物表達載體pGreen0029,得到ZmLTP3-dHA片段和表達載體,T4 DNA連接酶連接過夜,形成含有35S-C4DDPK-ZmLTP3-dHA-NOS片段的重組載體pGreen0029,并送至生工生物工程(上海)股份有限公司測序驗證。
1.2.2 ZmLTP3基因的轉化 將鑒定正確的質粒用凍融法轉化農桿菌GV3101,轉化后的農桿菌用花浸蘸法侵染擬南芥。
1.2.3 轉基因擬南芥的PCR檢測 轉基因擬南芥T0代的種子經消毒后播在含50 μg/mL卡那霉素的MS培養基上進行篩選,挑選在抗性培養基上正常生長的擬南芥植株提取基因組DNA,以DNA為模板,以ZmLTP3基因片斷內部和載體片段的1對特異引物進行PCR鑒定。
1.2.4 轉基因擬南芥的Western-blot檢測 取0.3 g經PCR鑒定呈陽性的擬南芥幼嫩的葉片,加0.5 mL蛋白提取緩沖液(10 mmol/L Tris-HCl,0.02% NaN3,0.001% PMSF,pH 8.0),液氮研磨后4 ℃、5 000 r/min離心10 min,吸取上清液,即得到總蛋白提取液。取適當總蛋白提取液電泳,轉膜后以小鼠單克隆抗體Anti-HA(3f10)為一抗,Peroxidase-conjugated goat anti-rat IgG (H+L) 為二抗,進行Western-blot 檢測。
2 結果與分析
2.1 ZmLTP3基因植物表達載體的構建
取BamHⅠ/PstⅠ酶切回收后的PCR片段和載體片段, 用T4 DNA連接酶連接,轉化大腸桿菌后,挑選陽性克隆提取質粒進行酶切驗證,所得的酶切片段與預期大小一致, 初步證明已經連接成功。將質粒進行測序, 做進一步驗證,結果確認了重組質粒構建成功。ZmLTP3重組質粒植物表達載體如圖1所示。
2.2 轉基因擬南芥的鑒定
經過測序鑒定正確的載體轉化農桿菌,攜帶表達載體的農桿菌介導轉化擬南芥, 轉化后收取T0代種子, 播種在含有50 μg/L卡那霉素的MS培養基上。種子發芽后大多數幼苗子葉變黃,只有少部分幼苗仍然發綠,并且正常長出真葉,保持正常的生長狀態,這部分很有可能就是轉化了的幼苗(圖2)。
為了排除抗生素篩選過程中的假陽性必須對初步篩選出的抗性植株做進一步的分子檢測。將在抗性培養基上正常生長的T1代幼苗轉移到土壤中,待植株充分長大,有足夠葉子時,取2~3片幼嫩的葉子提取基因組DNA,并以ZmLTP3基因與載體片段的特異引物進行PCR擴增,結果如圖3所示。野生型植株沒有擴增出相應條帶,同時也發現,抗性篩選出的幼苗也有假陽性。
為了進一步驗證轉基因株系,經過PCR 初步鑒定的株系,提取總蛋白,通過Western-blot進一步在蛋白水平檢測基因的表達,結果如圖4所示。除了野生型植株沒有印跡條帶外, 其他PCR 陽性的株系在13 ku左右處均有特異條帶。結果表明, 這些株系為轉基因株系,轉化的目的基因在植物中得到了表達,沒有出現基因沉默現象。
3 小結
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