小麦盐胁迫响应基因TaSRP的克隆及功能鉴定

doi:10.3864/j.issn.0578-1752.2014.12.002

摘要/Abstract

摘要： 【目的】土壤盐碱化是制约中国小麦生产的重要因素之一，耐盐性改良成为重要的育种目标。分析小麦TaSRP的功能，解析TaSRP的耐盐性作用机制。【方法】对小麦盐处理转录组测序结果进行分析，发现一个受盐胁迫诱导表达的小麦凝集素类基因TaSRP。根据TaSRP编码的氨基酸序列，通过在NCBI中比对得到水稻、大豆和拟南芥中与TaSRP相似的蛋白序列，利用DNAMAN和MEGA5.05软件进行多重序列比对和同源性分析。根据TaSRP的保守序列设计特异引物，利用TaSRP的实时荧光定量PCR检测TaSRP在不同胁迫处理条件下的表达模式；构建带有CaMV 35S启动子的16318hGFP-TaSRP绿色荧光表达载体，利用瞬时转染法将重组质粒和对照空载体导入拟南芥原生质体，室温黑暗培养16 h，激光共聚焦显微镜下观察绿色荧光信号；扩增TaSRP启动子序列，利用植物顺式作用元件数据库PLACE（http: //www.dna.affrc.go.jp/PLACE）分析启动子区域中参与非生物胁迫响应的顺式作用元件；将TaSRP的cDNA序列连入带有CaMV 35S启动子的pBI121表达载体中构建pBI121-TaSRP表达载体，转入C5C81农杆菌中，采用蘸花法侵染拟南芥得到转基因株系，并进行耐盐性鉴定。【结果】TaSRP具有EUL保守区，属于卫矛凝集素（EUL）家族，定位在细胞质内。氨基酸序列同源性及系统发育树分析发现，TaSRP与水稻的OSR40类蛋白（OSR40g3、OSR40g2和OSR40c1）的同源性最高；与拟南芥、大豆的同源性较低。TaSRP启动子含有一系列对盐、ABA和低温等非生物胁迫响应的调控元件。实时荧光定量PCR分析显示，TaSRP受ABA和盐胁迫上调表达，对干旱胁迫无明显响应。抗性试验结果显示，在不加NaCl的条件下，野生型与过表达株系总根长基本一致，在125 mmol•L-1 NaCl和150 mmol•L-1 NaCl处理的培养基上，3个转基因拟南芥株系的总根长均大于野生型拟南芥的总根长，并达到显著性差异，表明过表达株系有较好的耐盐性，TaSRP在拟南芥中过表达能够提高拟南芥对盐胁迫的抗性。【结论】TaSRP提高了转基因拟南芥对高盐的抗性。

关键词: 小麦（Triticum aestivum L.） , 卫矛凝集素家族 , 胁迫响应 , 实时荧光定量PCR , 耐盐性

Abstract: 【Objective】 Salt is one of the major constraints to yield and quality of wheat in China. How to improve salt tolerance of crops has become an important objective of breeding. The study provided experimental data for further study of the function and the mechanism of TaSRP gene. 【Method】 TaSRP was obtained by analysis of salt treatment wheat de novo transcriptome assembly. Homologous sequences of TaSRP in O. sativa, Arabidopsis and Glycine max were selected using the NCBI BLAST analysis program. Homology analysis and multiple alignments were performed with DNAMAN and MEGA5.05. Real-time PCR of TaSRP specific primers was used to analyze the expression patterns under various abiotic stresses. The coding region of TaSRP was fused to the N-terminal end of GFP under control of the CaMV 35S promoter as 16318hGFP-TaSRP. The 16318hGFP- TaSRP and control vectors were transformed into Arabidopsis protoplasts for subcellular localization. Results were visualized by a confocal microscopy after 16h at room temperature under dark conditions. The promoter region of TaSRP was amplified by PCR using specific primers. Cis-elements responding to abiotic stresses were analyzed by referring to the plant cis-element database PLACE （http: //www.dna.affrc.go.jp/PLACE）. Full length TaSRP cDNA was ligated into the vector pBI121 under control of the CaMV 35S promoter to construct an expression vectors for wild-type Arabidopsis plants. The pBI121-TaSRP construct was introduced into Agrobacterium tumefaciens C58C1 strain cells. Transgenic plants were used for salt resistant experiment. 【Result】 TaSRP, a ricin B-like lectin gene, was obtained by searching the results of salt treatment wheat de novo transcriptome assembly. TaSRP, containing a EUL domain, belongs to EUL family. Homology analysis and multiple alignments showed that there was a high degree of sequence similarity between TaSRP and OSR40 (OSR40g3, OSR40g2 and OSR40c1), but a low degree of sequence similarity between TaSRP and Arabidopsis and G. max. The subcellular localization assay indicated that TaSRP located in cytoplasm. RT-PCR analysis revealed that the expression of TaSRP was upregulated by ABA and salt, but not affected by drought. Isolation of the TaSRP promoter revealed a core promoter element and some cis-acting elements responding to abiotic stresses. Analysis of real-time quantitative PCR showed that TaSRP was up-regulated by high-salt and ABA stresses. The total root lengths of three TaSRP lines were significantly longer than those of wild-type plants in salt treatments (125 mmol•L-1 and 150 mmol•L-1 NaCl) in Arabidopsis plants. These results indicated that TaSRP lines improved salt tolerance of Arabidopsis. 【Conclusion】 TaSRP increased tolerance to salt stress of Arabidopsis plants.

Key words: wheat (Triticum aestivum L.) , EUL family , stress responses , real-time PCR , salt tolerance

胡笛1, 2, 徐兆师2, 崔晓玉2, 陈明2, 李连城2, 马有志2, 张小红1. 小麦盐胁迫响应基因TaSRP的克隆及功能鉴定[J]. 中国农业科学, 2014, 47(12): 2292-2299.

HU Di-1, 2 , XU Zhao-Shi-2, CUI Xiao-Yu-2, CHEN Ming-2, LI Lian-Cheng-2, MA You-Zhi-2, ZHANG Xiao-Hong-1. Isolation and Functional Analysis of Salt-Responsive Gene TaSRP in Wheat[J]. Scientia Agricultura Sinica, 2014, 47(12): 2292-2299.

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