野桑蠶核糖體蛋白L21基因的克隆及序列分析

翁梁

摘要：【目的】克隆野桑蠶（Bombyx mandaina）核糖體蛋白L21（RPL21）基因并進行半定量分析，為研究RPL21基因在昆蟲體內的生物學功能提供參考。【方法】采用RT-PCR克隆野桑蠶RPL21 基因序列，并采用半定量PCR分析該基因在野桑蠶4齡幼蟲不同組織中的表達情況。【結果】擴增獲得的野桑蠶RPL21基因序列全長為586 bp，其包含一個完整的開放閱讀框（ORF），長度為480 bp，編碼159個氨基酸；預測蛋白的分子量和等電點分別為18 kDa和11.01。序列比對結果表明，野桑蠶與其他12個物種的RPL21基因的相似性介于55.5%～91.6%，其中與致倦庫蚊（Culex quinquefasciatus）RPL21的相似性最低，與家蠶（Bombyx mori）的相似性最高。半定量分析結果表明，野桑蠶RPL21基因在野桑蠶幼蟲的中腸、絲腺、脂肪體、血液、睪丸、表皮、卵巢和腦組織中均有表達，且轉錄水平無明顯差異。【結論】野桑蠶RPL21基因進化較保守，在各組織中分布廣泛且表達水平穩定。

關鍵詞： 野桑蠶；核糖體蛋白L21；基因克隆；組織表達

0 Introduction

【Research significance】Bombyx mandarina Moore， a silk insect feed with leaves of mulkberry or paper mulkberry， belongs to the family bombycidae of Lepidoptera； it is the closest relative of Bombyx mori， and these two species were very similar in their morphology and physiological characteristics （Yukuhiro et al.， 2002）. The ribosomal protein is highly conserved in the process of biological evolution， and those found in eukaryotic cells were more than 80 species（Zhu et al.， 2010a）. It reported that a good deal of ribosomal proteins also had functions in composition of ribosome and protein biosynthesis， besides replication， transcription and processing， regulation and translation， DNA repair， drug-resistance formation， autologous translation， regulation of apoptosis and normal cells malignant transformation and developmental regulation and so on（Wool， 1992；Stoneley and Willis， 2003； Clemens， 2004）. Ribosomal protein L21（RPL21） is located at the 60S subunit of eukarya， its specific functions are still not explained very good so far. Therefore， the cloning and semi-quantitative analysis of RPL21 gene in different species could make us well know about its distribution and expression， which would provide a molecular basis for future research.【Research progress】In recent years， RPL21 gene of some plant species viz.， wheat， Arabidopsis and Hevea brasiliensis， etc. have been cloned and further studied（Portereiko et al.， 2006； Li et al.， 2011； Zou and Yang， 2011）. The RPL21 gene of wheat had cDNA of 521 bp encoding 271 deduced amino acids， and could be highly expressed in vigorous growth tissues（Li et al.， 2011）. The results reported by Zou and Yang（2011） indicated that， the RPL21 gene of Hevea brasiliensis Muell. Arg. had cDNA of 853 bp encoding 271 deduced amino acids； it was related to tapping panel dryness syndrome and showed a decreasing expression level in tapping panel dryness. As to insects， the RPL21 gene was also cloned from silkworm， Aedes aegypti and Tribolium castaneum（Nene et al.， 2007； Diez-Roux et al.， 2011； Suetsugu et al.， 2013）， etc. Suetsugu et al.（2013） found that the RPL21 gene of silkworm encoded a 18 kDa of protein， which was accordance with the results of swallowtail butterfly（Papilio xuthus） reported by Futahashi et al.（2012）.【Research breakthrough point】Till to now， the immunogenes viz.， antivirus protein genes， cytochrome P50 family genes were the main researching aspects in B. mandarina（Yao et al.， 2008； Wang et al.， 2009； Zhao et al.， 2010）， but related researches on RPL12 gene is still reported rarely. 【Solving problems】In the present study， the RPL21 gene was cloned from B. mandarina， followed by sequencing and expressing analysis in different tissues by using semi-quantitative ana-

lysis， which would provide references for researching its biological functions in insect.

1 Materials and methods

1. 1 Experimental insects

The 2-3 instar larva of B. mandarina were co-

llected from mulberry tree and reared with fresh mulberry leaves indoor under 25 °C with relative humidity 70%-80%. The midgut， silk gland， fat body， hemocytes， testis， integument， ovary and brain were dissected from fourth instar larvae on the 3rd day and stored at -80 °C until to be used for semi-quantitative PCR analysis.

1. 2 Experimental methods

1. 2. 1 Total RNA extraction of fat body and cDNA synthesis The 100 mg of larvae fat body was grinded with liquid nitrogen to extract total RNA by using TRIzol method（Zhu et al.， 2010a） with RNAiso Plus（TaKaRa， Dalian， China） according to the protocol. The quantity and purity of the extracted RNA were quantified by using NanoDrop 1000 spectrophotometer（NanoDrop Technologies Wilmington， DE） and agarose gel electrophoresis， respectively； followed by storing at -80 ℃. The purified RNA（1.0 μg） was used as template to synthesize first strand cDNA by using M-MLV reverse transcriptase（TaKaRa， Dalian， China） following the manufacturers instructions.

1. 2. 2 Cloning and sequencing of BmRPL21 gene

According to the conversed region sequences of RPL21 gene in different species， the primers RC5（5'-GTGTGGCATACCCTTTTGAA-3'） and RC3（5'-TGGAG

ACATTGTAGACATCAGAGG-3'） for amplifying RPL21 gene， and F1（5'-GAACTATCCCGCTCTCCAC-3'） and R1（5'-GACGGGTTTCTCAGTTCC-3'） for semi-quantitative analysis were designed with Primer premier 5.0 software package and synthetized by Sangon Biotech（Shanghai， China）.

The synthetize cDNA was used as template to conduct PCR amplification. The 25.0 μL PCR reaction system was composed of 1.0 μL cDNA， 2.5 μL 10×PCR Buffer， 2.0 μL dNTP（2.5 mmol/L）， 1.0 μL 10 μmol/L of each primer， 17.2 μL sterilized ddH2O and 0.3 μL Taq polymerase. The PCR amplified program were done under the following conditions： initial denaturation for 4 min at 94 ℃； followed by 35 cycles of denaturing for 30 s at 94 ℃， annealing for 30 s at 56 ℃， and extending for 40 s at 72 ℃； with a subsequent 10 min for final extension at 72 ℃.

The 5.0 μL PCR products were fractionated by agarose gel electrophoresis（1% w/v），purified and recycled by using a DNA gel extraction kit（Aidlab， Beijing， China）. Then the purified PCR products were ligated into pMD19-T vector（TaKaRa， Dalian， China）， and transferred into competent cell DH5α for culture. The positive recombinant clone was sequenced at least three times by Sangon Biotech（Shanghai， China）.

1. 2. 3 Bioinformatics analysis of BmRPL21 gene

The sequence was analyzed by DNASTAR software package. In addition， the sequence was performed using the online BLAST tools in NCBI web site（http：//blast.ncbi.nlm.nih.gov/Blast）. Multiple sequence alignment was carried out using ClustalW software. The ORF was searched by ORF Finder from NCBI. Phylogenetic tree was constructed by MEGA version 5.0 using the neighbor-joining（NJ） method with bootstrap test of 1000 replications.

1. 2. 4 Tissue expression analysis of BmRPL21 gene

The RNA of midgut， silk gland， fat body， hemocytes， testis， integument， ovary and brain were extracted by using TRIzol method and conducted to reverse transcription into cDNA. The cDNA samples were diluted to 100 ng/μL with RNase-free water and employed as templates in semi-quantitative PCR analysis.

The expression levels of BmRPL21 mRNA in different tissues were examined by a semi-quantitative PCR. The primers F1（5'-GAACTATCCCGCTCTCCA

C-3'） and R1（5'-GACGGGTTTCTCAGTTCC-3'） were used to amplify a product of 270 bp. The 18S rRNA gene was selected as the reference to standardize normalization， its primer and sequences were as follows： F18S（5'-GTAGTAGACAATGGCTCC-3'） and R18S（5'-AGACTCGTCGTACTCCTG TT-3'）. The semi-quantitative PCR was performed in a total volume of 25.0 μL containing 12.5 μL 2×PCR Mix（TaKaRa， Dalian， China）， 1.0 μL forward and reverse primers， 1.0 μL cDNA， and 9.5 μL RNase-free H2O. The QRT-PCR was conducted under the following conditions： initial denaturation for 4 min at 94 ℃， followed by 30 cycles of denaturation for 30 s at 94 ℃， annealing for 30 s at 57 ℃， and extension for 20 s at 72 ℃. The PCR products were fractionated by agarose gel electrophoresis（1% w/v）.

2 Results and analysis

2. 1 PCR amplification of RPL21 gene

After amplified with primers RC5 and RC3， and F1R1， three amplified fragments in length of 200， 480 and 270 bp were obtained（Fig.1），which were accordance with expectation， and then assembled using the SeqMan II program from the Lasergene software pac-

kage（DNASTAR Inc. Madison， USA）.

2. 2 Sequence analysis of RPL21 gene

The sequencing analysis results revealed that， the full-length cDNA of BmRPL21 was 586 bp， contained a 60 bp 5'-untranslated sequence（5'-UTR）， a 46 bp 3'-untranslated region（3'-UTR） and an open reading frame（ORF） of 480 bp encoding a putative protein of 159 amino acid residues（Fig.2）. The theoretical molecular weight of 18 kDa and isolectric point（pI） of 11.01 were found in L21 protein.

2. 3 Expression analysis of Bm-RPL21 gene in di-

fferent tissues of B. mandaina

From the analysis results of semi-quantitative PCR for midgut， silk gland， fat body， hemocytes， testis， integument， ovary and brain of B. mandaina， we found that the BmRPL21 gene was expressed in all above-mentioned tissues， and the level of BmRPL21 transcription was steady without obvious difference（Fig.5）.

3 Discussion

There are various kinds of ribosomal protein species in organism， with regulation of gene transcription， apoptosis and immune response involved in ribose and other functions in vitro（Warner and Mclntosh et al.， 2009）. Currently， the RPL21 gene registered in GenBank are more than one thousand， which were mainly derived from insects and microorganisms， as well as a small number of plants， but great differences of RPL21 gene sequence were found between plants and insects. Although more RPL21 gene sequence resources have been reported， less in-depth studies on RPL21 gene were conducted， especially for its function verification. Previous researches indicated that， the mutations of RPL21 gene from Arabidopsis could result in the development and fertilization hindered of female gametes， maybe it was involved in nuclear fusion（Portereiko et al.， 2006）. The transcription level of RPL21 gene from Hevea brasiliensis was down regulated in the “dead” rubber tree latex， which indicated that the activity of metabolism in mitochondrion and even in entire latex cells maybe down regulated（Zou and Yang et al.， 2011）.

At present， in-depth studies of ribosomal protein genes in insects were mainly on L7， S3a and other genes（Zhu et al.， 2010a， 2010b；He et al.， 2008）， but rare on RPL21. The ribosomal protein gene S3a was cloned from Antheraea pernyi and had a 795 bp of ORF encoding 264 deduced amino acids； S3a protein in Antheraea pernyi had 72%-99% of similarity with other species； S3a could be expressed in different ti-

ssues of Antheraea pernyi（Zhu et al.， 2010a）. B. mandarina is widely perched on Chinese sericulture area and found in the silkworm regions of Japan， North Korea， South Korea and other countries. In the present study， the RPL21 gene of B. mandaina was obtained by subcloning method. With a full-length of 586 bp， it was similar with Papilio polytes RPL21（573 bp）， but longer than the following ones： B. mori RPL21（550 bp）， Papilio xuthus RPL21（552 bp）， Spodoptera frugiperda RPL21（562 bp） and other species（Landais et al.， 2003）， and shorter than Helicoverpa zea RPL21（788 bp）， Mus musculus RPL21（1693 bp） and other species（Kondrashov et al.， 2011）. The deduced amino acid sequence encoded by B. mandaina RPL21 gene shared a highest homology with that of B. mori in previous report， which further indicated the close relationship between B. mori and B. mandaina， and confirmed the conclusion that B. mori was originated from B. mandaina（Yukuhiro et al.， 2002）. From the constructed evolutionary tree results of amino acid sequences in ribosomal protein L21 between B. mandaina and the other 12 species， there existed a remarkable branch among lepidopteran， dipteran insects and vertebrates， but insignificant difference was found between similar species， showing that structure specificity of RPL21 gene presented in different species. Besides， the amino acids of RPL21 protein in different species showed a high coherence in certain regions， which demonstrated that RPL21 gene was conservative during its evolution. Thus， it is supposed that RPL21 gene may be has some same or similar functions in different organisms belonging different species. In addition， there is a remarkable phenomenon that a very large differences were found in the number and types of terminal amino acids of RPL21 among different species. This result was somewhat similar with the research results of S3a and other genes， but what is the reason for this difference formed， and whether it can be the reference mark of genetic classification of not， which are needed to be in-depth studied in the future. The RT-PCR results showed that， RPL21 gene detected in different tissues of in B. mandaina 4th instar larvae was stably expressed without little difference， which may be related to broad-based biological functions（Lindstrom， 2009）， possibly due to RPL21 gene extensively involved in the biosynthesis of proteins in different organizations. However， the specific functions of RPL21 gene in B. mandaina still need to be further studied.

4 Conclusion

The results showed that the BmRPL21 gene could be widely expressed in different tissues of B. mandaina without significant difference in transcriptional level； BmRPL21 gene had the nearest evolutionary relationship to B. mori.

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