番茄抗性相关基因SlHin1的克隆、表达与抗病毒功能

doi:10.3864/j.issn.0578-1752.2017.07.007

摘要/Abstract

摘要： 【目的】克隆番茄抗性相关基因SlHin1（harpin-induced gene 1），分析其生物信息学特征、组织表达和亚细胞定位情况，研究其在抗烟草花叶病毒侵染、移动中的作用，为番茄抗性育种提供理论依据。【方法】通过RT-PCR及基因克隆方法，从番茄总RNA中克隆得到基因SlHin1；应用生物信息学方法分析其DNA序列及其编码的蛋白序列特性，使用MEGA6.0对SlHIN1氨基酸序列及其同源序列进行多序列比对，并构建同源物种间系统进化树；将该基因片段通过Sal I和Bam HI双酶切连接至荧光表达载体pCV-mGFP-C1，采用GFP标记的方法进行亚细胞定位检测，分析编码蛋白表达位置；利用实时荧光定量RT-PCR（qRT-PCR）分析该基因在番茄不同组织的表达量水平；将该基因片段通过Nco I和Bam HI通过双酶切连接至植物表达载体pFGC5941，用土壤农杆菌EHA105瞬时过表达该基因并接种标记有绿色荧光的烟草花叶病毒侵染性克隆，以检测植株对病毒的抗性变化，间接ELISA法检测病毒的含量，探究该基因的抗病毒功能。Western blot验证SlHIN1蛋白在本氏烟内的表达情况。【结果】利用RT-PCR方法从番茄材料（Solanum lycopersicon cv. Ailsa Craig）的叶片组织中克隆得到全长为675 bp的番茄抗性相关基因SlHin1，将序列分别提交至GenBank，得到序列号KU195820。序列比对及生物信息学分析表明，其基因预测编码225个氨基酸，分子量为26.1 kD，理论等电点为9.35，结构上具有LEA-14保守结构域，不具有跨膜区段，并且位于番茄基因组10号染色体上（Solyc10g081980）；多序列比对及进化树分析表明，该基因编码的蛋白与茄科植物HIN1氨基酸同源性80.0％以上而与水稻、高粱单子叶植物的亲缘关系较远；亚细胞定位显示定位在细胞膜上与PSORT Prediction预测的亚细胞定位最高概率一致；qRT-PCR结果分析表明该基因具有组织特异性，表达量在根、叶、茎间依次降低；在本氏烟中瞬时过表达SlHIN1并在表达部位接种标记有绿色荧光的烟草花叶病毒侵染性克隆，处理组本氏烟在接种病毒4 d后没有任何荧光出现，而对照组出现绿色荧光。随着接种时间推移，接种7 d后处理组叶片上零星荧光有略微的扩大，而对照组的绿色荧光已经扩散至心叶。间接ELISA检测病毒含量较对照组少，处理组的病毒扩散受到抑制。Western blot分析结果表明，显色后的硝酸纤维素膜上约在26 kD处有一特异条带，而空载体的对照无相应条带，说明SlHIN1蛋白在注射部位成功表达。【结论】Hin1在供试科植物中均存在，其中SlHin1定位在细胞膜，过表达该基因可抑制病毒扩散，推测其可能参与茄科植物对烟草花叶病毒的抗性反应，使植株获得抗性。

关键词: 番茄, SlHin1, 烟草花叶病毒, 表达, 抗病毒

Abstract: 【Objective】The objective of this study is to clone tomato resistance-related gene SlHin1, to analyze its bioinformatics characters, tissue expression and subcellular localization, evaluate its anti-role in the process of the infection and movement of Tobacco mosaic virus (TMV), and to provide a theoretical basis for tomato resistance breeding. 【Method】 Gene cloning and RT-PCR were combined to isolate a gene which might encode SlHIN1. Bioinformatics tools were applied to analyze both the DNA sequence and the characteristics of encoded protein sequence, MEGA6.0 was used to make multiple sequence alignments between the SlHIN1 amino acid sequences and their homologous sequences, and the phylogenetic tree of homologous species was constructed. The subcellular localization test of SlHIN1 was analyzed by using fusion expression vector pCV-mGFP-C1, then the gene was inserted into the expression vector by Sal I and Bam H I digestion. The real-time fluorescent quantitative RT-PCR (qRT-PCR) was used to analyze to the expression levels of SlHin1 in different tomato tissues. The gene fragment was inserted into the plant expression vector pFGC5941 by Nco I and Bam HI digestion. The SlHIN1 was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the SlHIN1 expressed leaves were inoculated with TMV-GFP. The accumulation of the virions was detected by indirect ELISA to investigate the antiviral function of the gene. Western blot was used to evaluate the expression of SlHIN1 protein.【Result】Using RT-PCR method, the tomato Hin1 was obtained from tomato (Solanum lycopersicon cv. Ailsa Craig) leaves and was designated as SlHin1 (GenBank number: KU195820). SlHin1 was 675 bp in length. It was predicted to encode a protein with 225 amino acid residues, a molecular weight of 26.1 kD and a theoretical isoelectric point of 9.35. SlHin1 contains the LEA-14 domains structure, doesn’t have transmembrane segments and locates on chromosome 10 (Solyc10g081980). Sequence analysis and phylogenetic tree analysis showed that SlHIN1 shared approximately 80% similarity with HIN1 from other solanaceae plants and was close to that of rice and sorghum monocotyledons. Subcellular localization was showed that SlHIN1 distributed on the plasma membrane of the leaf epidermis cell of N. benthamiana, which is consistent with the predicted results. The results of qRT-PCR showed that the SlHin1 was of tissue-specificity, whose expression decreased from tomato roots, leaves to stems. The SlHIN1 was transiently expressed in the N. benthamiana leaves by agro-infiltration, and the SlHIN1 expressed leaves were inoculated with TMV-GFP. After 4 days, there was no green fluorescence observed in the SlHIN1 expressed leaves under UV light, but the green fluorescence could be observed in the control group. With the passage of the inoculation, the sporadic fluorescence on the leaves of the treated group was slightly enlarged after 7 days post inoculation, and the green fluorescence of the control group had spread to the leaf. The spread of virus was inhibited and the time that the virus needed to reach the lobus cardiacus was longer, the content of the virions was less than that of control group through indirect ELISA test result. Western blot analysis showed that there was a specific band at about 26 kD in the nitrocellulose membrane, and no corresponding control was observed in the empty vector, indicating that the SlHIN1 protein was successfully expressed at the injection site.【Conclusion】Hin1 genes are present in all tested species, and the SlHIN1 is localized on the plasma membrane of the leaf epidermis cell of N. benthamiana. Transiently expressing SlHIN1 could inhibit the accumulation of the virions and spread of the virus, which can reflect that it may be involved in the resistance reaction of Solanaceae plants to TMV causing them to have resistance.

Key words: tomato, SlHin1, Tobacco mosaic virus (TMV), expression, anti-virus

彭浩然，潘琪，魏周玲，蒲运丹，张永至，吴根土，青玲，孙现超. 番茄抗性相关基因SlHin1的克隆、表达与抗病毒功能[J]. 中国农业科学, 2017, 50(7): 1242-1251.

PENG HaoRan, PAN Qi, WEI ZhouLing, PU YunDan, ZHANG YongZhi, WU GenTu, QING Ling, SUN XianChao. Cloning, Expression and Anti-Virus Function Analysis of Tomato Resistance-Related Gene SlHin1[J]. Scientia Agricultura Sinica, 2017, 50(7): 1242-1251.

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