番茄SlGRAS4基因特征分析和耐热功能鉴定

Identification and Heat Resistance Analysis of SlGRAS4 Gene in Tomato

GRAS转录因子是调控植物生长发育和非生物胁迫响应的重要转录因子之一,而目前还没有GRAS调控高温胁迫的研究。为了深入研究番茄SlGRAS4生物功能,以耐热番茄LA2093为试验材料,分析番茄SlGRAS4基因结构、启动子序列及进化关系,利用qRT-PCR检测SlGRAS4在不同胁迫和不同激素处理下的表达水平,利用VIGS验证SlGRAS4基因耐热功能。结果表明:(1)生物信息学分析显示,SlGRAS4蛋白长度为666 aa,分子量为75737.72 Da,理论等电点为6.31,含有GRAS转录因子家族典型的结构域,主要集中在C末端的277~657 aa之间;在SlGRAS4启动子区域发现脱落酸(ABA)和水杨酸(SA)响应元件;SlGRAS4与烟草NtGRAS1蛋白亲缘关系最近,推测SlGRAS4可能与其同源基因具有相似的生物功能。(2)在高温、低温、盐和干旱胁迫处理12 h时番茄SlGRAS4基因表达量升至最高,分别增加到对照的8.86、4.86、55.38和7.63倍;在ABA和SA激素处理8 h时SlGRAS4基因的表达量达到峰值,分别达到对照的120.72和3.55倍,说明SlGRAS4可能参与了多种非生物胁迫响应和激素信号传导。(3)沉默SlGRAS4基因番茄植株(VSlGRAS4)在高温胁迫下较对照植株(Ve)更容易萎蔫,且Fv/Fm与SOD、POD活性显著降低,REL和H_(2)O_(2)含量显著升高,说明在高温胁迫下沉默SlGRAS4使番茄植株细胞膜氧化损伤加重,光合能力降低,活性氧(ROS)清除酶活性减弱。(4)qRT-PCR分析显示,VSlGRAS4植株中高温信号应答关键基因HsfA1b、ROS信号应答基因ZAT10和ZAT12以及ROS清除酶编码基因CuZnSOD、FeSOD、APX1、APX2、CAT的表达水平均显著低于Ve植株,表明SlGRAS4转录因子可以通过调控高温和ROS信号转导来影响番茄的耐热性。研究认为,高温、低温、干旱、盐、ABA和SA均可显著诱导番茄SlGRAS4基因的表达,沉默SlGRAS4基因番茄植株的耐热性显著降低,证明番茄SlGRAS4基因具有耐热功能,为进一步解析SlGRAS4参与番茄耐热调控的分子机制奠定基础。

GRAS transcription factor is one of the most important transcription factors for plants in regulating growth and abiotic stress response.However,there have no research about GRAS regulating high temperature stress.In order to further explore the functions of SlGRAS4 gene in tomato,we used heat resistant tomato LA2093 as the test material,identified the gene structure,promoter sequence and evolutionary relationship of SlGRAS4 gene,detected the expression level of SlGRAS4 gene upon abiotic stress and hormone treatments by qRT-PCR,analyzed the function of SlGRAS4 gene responding to high-temperature by VIGS.The results showed that:(1)bioinformatics analysis showed that the length of SlGRAS4 protein was 666 amino acids,with 75737.72 Da molecular weight and 6.31 isoelectric point,the typical domain of the GRAS family was specifically detected at the C-terminal between 277-657 aa in SlGRAS4 protein;the elements related to abscisic acid and salicylic acid responding were detected in promoter region of SlGRAS4 gene;evolutionary relationship analysis showed SlGRAS4 protein has the nearest relationship with Nicotiana tabacum NTGRAS1 protein,and predicted SlGRAS4 has similar biological functions with its homologous genes.(2)The expression of SlGRAS4 gene in tomato increased to the highest and up-regulated 8.86,4.86,55.38 and 7.63 folds compared with the control after 12 h of high temperature,low temperature,salt and drought stress treatments,respectively;as well as reached the peak value after 8 h of ABA and SA treatments,which up-regulated 120.72 and 3.55 folds compared with the control,respectively,indicating that SlGRAS4 may be involved in a variety of abiotic stress responses and hormone signal transduction.(3)Under high temperature stress,SlGRAS4-silencing tomato plants(VSlGRAS4)were wither than the control plants(Ve),and Fv/Fm,SOD and POD activities were significantly reduced,and contents of REL and H_(2)O_(2) were significantly increased,indicating that silencing SlGRAS4 aggravated oxidative damage of the cell membrane,and reduced photosynthetic capacity,and weakened activity of ROS scavenging enzyme in tomato plants.(4)qRT-PCR analysis showed that the expression level of HsfA1b(high temperature signal response gene),ZAT10 and ZAT12(ROS signaling response genes),CuZnSOD,FeSOD,APX1,APX2 and CAT(ROS scavenging enzyme code genes)were also significantly down-regulated in VSlGRAS4 plants.The results indicated that SlGRAS4 can enhance heat tolerance of tomato through high-temperature and ROS signal transduction.The studies showed the expression of SlGRAS4 gene was significantly induced under high temperature,low temperature,drought,salt,ABA and SA,and the heat tolerance of tomato was reduced after silencing SlGRAS4 gene,indicating tomato SlGRAS4 gene has heat resistance function,and this will lay a foundation for further analysis of heat molecular mechanism.