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

【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。

Cloning and Function Analysis of Heat-Shock-Protein LsHsp70-2711 Gene Under High Temperature Stress in Leaf Lettuce (Lactuca sativa L.)

【Objective】Through cloning of genes related to Hsp70, and using VIGS to analyze Hsp70 expression and morphological change of leaf lettuce under heat stress to lay a foundation for analysis of the response mechanism and molecular mechanism of heat shock protein under heat stress.【Method】The full-length cDNA gene of LsHsp70-2711 gene was obtained by homologous cloning and RACE technology. By real-time fluorescence quantitative PCR (qRT-PCR), the expression of the gene from P-S11and G-S59 was analyzed. According to the VIGS technology, the pTRV-LsHsp702711 vector was constructed and transformed into Agrobacterium GV1301. The leaves of lettuce were infected by the injection method. After three weeks, the positive plants were identified by PCR. The gene expression and morphology were compared between the control group and positive group, and the expression characteristics of LsHsp70-2711 were analyzed again after heat stress and drought treatment. 【Result】The sequence analysis indicated that the full-length cDNA was 2226 bp, the open reading frame was 2154 BP, encoding 718 amino acids, and its homology compared with that of Arabidopsis (NP_187864.1), Tomato (NP_001266213.1), Saussurea Medusa (AAB99745.1), and other species. qRT-PCR results showed that the expression of the gene in the two cultivars was up-regulated, and the expression level of heat resistant cultivars was significantly higher than the heat sensitive cultivar, and the gene expression of P-S11 was inhibited by the increase of stress time, and the expression level of G-S59 was higher in the heat resistant variety. In heat resistant variety, the maximum expression of LsHsp70-2711 appeared at 42℃, 60 min, and the maximum expression of heat sensitive variety reached at 37℃, 60 min. Prediction of subcellular localization showed that LsHsp70-2711 was mainly in the cytoplasm. The constructed vector was used to invade the leaves of lettuce, and the positive plants were obtained after identification. LsHsp70-2711 expression decreased after it was silenced by VIGS, compared with the control, the stem length of the positive plants was significantly increased. After heat stress and drought treatment, the expression of LsHsp70-2711 was significantly lower than the control, and the influence of high temperature treatment on LsHsp70-2711 was greater than drought stress.【Conclusion】 LsHsp70-2711 gene belongs to Hsp70 gene family, and is related to the heat resistance in lettuce. The research results of the study will provide theoretical supports for the analysis of function of high temperature in bolting of lettuce.