玉米转录因子WRKY76克隆及抗纹枯病表达模式分析

Cloning of maize WRKY76 and its expression patterns involved in resistance to maize banded sheath blight

关键词:  纹枯病, 转录调控因子, WRKY基因, 实时荧光定量PCR

Key words:  maize banded leaf and sheath blight, transcription factor, WRKY76, qRT-PCR

摘要:

玉米纹枯病是影响玉米产量和品质的重要病害之一。转录因子WRKY家族部分成员能够调控水杨酸和茉莉酸甲酯信号传递方式来激发防卫反应基因的表达。在NCBI上检索玉米中WRKY家族成员及拟南芥中抗病相关的WRKY家族成员,利用CLUSTAL X和MEGA5.05构建系统进化树,发现转录因子WRKY76可能参与玉米抗纹枯病的调控途径。该研究以玉米抗纹枯病材料R15和感病材料Ye478为对象,在玉米拔节期接种立枯丝核菌AG1-IA,首先分别于接菌前(对照)和接菌后1、2、4、6、12、24 h取叶鞘; 然后分别进行水杨酸和茉莉酸甲酯胁迫处理,分别于处理前(对照)和处理后1、2、4、6、12 h取叶鞘,提取RNA,实时荧光定量PCR分析WRKY76转录因子基因在玉米叶鞘组织中不同胁迫条件下的差异表达。结果表明:在立枯丝核菌AG1-IA胁迫下,WRKY76转录因子基因在胁迫后1 h表达量达最大值,抗病材料R15的相对表达量高于感病材料Ye478且差异显著(P≤0.05); 经水杨酸(Salicylic Acid, SA)处理,WRKY76在抗感材料中表达趋势相似,在感病材料掖478中,WRKY76被诱导而显著地上调表达,在抗病材料中,相对表达量峰值出现在胁迫后4 h,且相对表达量低于感病材料掖478。经茉莉酸甲酯(Methyl jasmine, MeJA)处理,WRKY76基因在感病材料中呈现下调表达趋势。WRKY76基因在1 h表达量为对照的0.6倍,其他调查时间点基本都在0.1～0.3之间。在抗病材料R15中,WRKY76基因表达呈现上升趋势,变化趋势不明显。这表明WRKY76转录因子基因能够被病原物、SA、MeJA诱导表达,可能参与植物抗纹枯病调控途径。

Abstract:

Maize banded leaf and sheath blight(BLSB)which is a very destructive disease, can result in a significant yield loss and also heavily affect the quality of corn in maize production area. The expression of defense response genes could be stimulated by SA(salicylic acid)and MeJA(methyl jasmine)in the signal transduction pathway, and regulated by members of the WRKY transcription factors family. Transcription factors WRKY family members in Maize and WRKY family members which are related to resistance in Arabidopsis were collected from NCBI, and then polygenetic tree was successfully constructed. According to the result of polygenetic which was conducted by using the software CLUSTAL X and the software MEGA5.05, we concluded that the transcription factor WRKY76 was most likely involved in maize sheath blight resistance network. Whether the transcription factor WRKY76 plays a truly important role in sigjnal transduction path is still unknown. The materials used in this study were R15(Resistant line)and Ye478(Susceptible line). R15 and Ye478 under different kinds of stress conditions were used to characterize the expression pattern of WRKY76 gene by qRT-PCR, leaf sheath samples were collected at 0, 1, 2, 4, 6, 12 and 24 h post Rhizoctonia solani AG1-IA inoculation, and 0, 1, 2, 4, 6, 12 h after SA(salicylic acid)and MeJA(methyl jasmine)stress treatment. When leaf sheath samples were inoculated with R. solani AG1-IA at jointing stage, the WRKY76 gene expression was able to reach to the highest at 1 h post inoculation, and its expression was 20 and 32 times higher than control in Ye478(Susceptible line)and R15(Resistant line), with significant difference(P<0.05). The relative expression decreased at 2 h post inoculation. 3 times for Ye478(Susceptible line)while it returned to the original level for R15(Resistant line). The expression pattern in R15(Resistant line)tended to increase along with treatment time increasing, while it was always higher than the susceptible line Ye478 with significant difference(P<0.05). When the maize leaf sheath was treated with SA(salicylic acid), the expression pattern of WRKY76 between Ye478(Susceptible line)and R15(Resistant line)were almost the same level, and its expression in R15(Resistant line)was higher than that of Ye478(Susceptible line). For R15(Resistant line), the relative expression level of WRKY76 was 33 times at 1 h post inoculation compared to the control, 46 times at 2 h post inoculation, and then decreasd to 2.5 time at 12 h post inoculation. However, When the maize leaf sheath was treated with MeJA, Ye478(Susceptible line)presents the downward trend, R15(Resistant line)shows a upward pattern according to the results of relative expression level at each treatment time. There was no significant difference between Ye478(Susceptible line)and R15(Resistant line). Finally research results of this study indicated that the WRKY76 gene expression could be induced by R. solani AG1-IA, SA(salicylic acid)and MeJA(methyl jasmine), and the WRKY76 might be involved in plant resistance signal transduction regulation pathway to maize banded leaf and sheath blight.