叶用莴苣热激蛋白基因LsHsp70-2711的克隆及高温胁迫下的功能分析

发布时间：2018-03-17 08:25

  本文选题：叶用莴苣　切入点：Hsp　出处：《中国农业科学》2017年08期 　论文类型：期刊论文

【摘要】：【目的】通过克隆Hsp70相关基因,并利用VIGS分析叶用莴苣热胁迫下Hsp70表达量和形态变化,为解析热激蛋白基因Hsp70在高温胁迫下的响应机制及分子机理奠定基础。【方法】通过同源克隆及RACE技术,获得叶用莴苣热激蛋白LsHsp70-2711的cDNA全长序列,利用实时荧光定量PCR(qRT-PCR)分析该基因在不同温度和不同高温时间下的叶用莴苣热敏品种‘P-S11’和耐热品种‘G-S59’的表达差异,确定基因和高温的相关性。根据VIGS技术,构建p TRV-LsHsp702711瞬时沉默载体,转化农杆菌GV1301,注射法侵染叶用莴苣叶片,三周后经PCR鉴定得到阳性植株。对照组和阳性组在基因表达和形态上进行对比,对照植株和阳性植株热胁迫和干旱处理后再次分析LsHsp70-2711的表达特性,观测形态变化。【结果】LsHsp70-2711 cDNA全长为2 226 bp,开放阅读框为2 154 bp,编码718个氨基酸,与拟南芥(NP_187864.1)、番茄(NP_001266213.1)、水母雪莲花(AAB99745.1)等物种的Hsp70同源性达到80%以上,证明此基因属于Hsp70家族。根据qRT-PCR结果,高温胁迫下该基因在两个品种中的表达均上调,在耐热品种中总体表达水平均高于热敏品种,耐热品种LsHsp70-2711的表达量最大值出现在42℃、60 min,37℃、60 min时热敏品种基因表达量达最大值,且在42℃高温下热敏品种‘P-S11’中基因表达随着胁迫时间的增加受到抑制,而耐热品种‘G-S59’则能长时间保持较高的表达水平,此结果和田间两品种间耐热差异表现相符合。亚细胞定位显示,LsHsp70-2711主要在细胞质中。将构建好的载体侵入叶用莴苣,鉴定后获得阳性植株。由定量PCR结果可知,未进行胁迫处理的阳性植株与对照株相比,LsHsp70-2711表达量下降,茎长明显增长。热胁迫和干旱处理后的阳性植株LsHsp70-2711表达量显著低于对照植株,高温处理对LsHsp70-2711的影响大于干旱胁迫。【结论】LsHsp70-2711属于Hsp70基因家族,其与叶用莴苣耐热性相关。研究结果为解析叶用莴苣热激蛋白LsHsp70-2711在叶用莴苣高温抽薹方面的功能提供了理论支持。
[Abstract]:[objective] to clone Hsp70 related genes and analyze the changes of Hsp70 expression and morphology under heat stress in lettuce by VIGS. In order to elucidate the response mechanism and molecular mechanism of heat shock protein gene Hsp70 under high temperature stress. [methods] the full-length cDNA sequence of heat shock protein LsHsp70-2711 in lettuce was obtained by homologous cloning and RACE technique. Using real-time fluorescence quantitative PCR qRT-PCR to analyze the difference of expression of the gene in heat sensitive variety of lettuce under different temperature and time, P-S11 'and heat-resistant variety GS59', to determine the correlation between gene and high temperature. The transient silencing vector of p TRV-LsHsp702711 was constructed and transformed into Agrobacterium tumefaciens GV1301. The lettuce leaves were infected by injection method. The positive plants were identified by PCR three weeks later. The gene expression and morphology of the control group and the positive group were compared. After heat stress and drought treatment in control and positive plants, the expression of LsHsp70-2711 was analyzed again, and morphological changes were observed. [results] the total length of LsHsp70-2711 cDNA was 2 226 BP, the open reading frame was 2 154 BP, encoding 718 amino acids. The homology of Hsp70 was more than 80% with other species such as Arabidopsis thaliana 187864.1, Tomato NP001266213.1 and AAB99745.1), which proved that this gene belonged to the Hsp70 family. According to the results of qRT-PCR, the expression of this gene in both cultivars was up-regulated under high temperature stress. The total expression level of heat resistant varieties was higher than that of heat sensitive varieties, and the maximum expression of LsHsp70-2711 in heat resistant varieties reached the maximum at 42 鈩，

本文编号：1623933