甘蓝型油菜谷胱甘肽转移酶基因BnGSTU12抗菌核病功能分析

Stem rot caused by Sclerotinia sclerotiorum was one of the most serious diseases, which can result in major losses in yield and quality. Genome-wide association analysis (GWAS) of 347 accessions was performed using the Illumina 60K Brassica SNP array. The detached stem inoculation assay was used to select five highly resistant (R) and susceptible (S) B. napus lines, 48 h post inoculation with S. sclerotiorum for transcriptome sequencing. A glutathione-S-transferase (GST) gene cluster (BnaC06g31020D, BnaC06g31030D and BnaC06g31040D) was found combining genome-wide association analysis (GWAS) and RNA-seq. Glutathione (GSH) pathway was activated after B. napus was infected with S. sclerotiorum. In addition, candidate gene association analysis with the re-sequencing data of B. napus in our group was performed, and BnGSTU12 was identified as the candidate gene. In this project, we will investigate the function of BnGSTU12, explore the mechanism of disease resistance and develop resistance marker for molecular marker assisted breeding. This study is helpful for the resistance breeding in B. napus.

菌核病是由核盘菌侵染植物引起的一种重要病害，可以造成油菜产量降低和品质下降。前期我们对甘蓝型油菜自然群体菌核病抗性进行了全基因组关联分析，并对核盘菌接种前后的极端抗感材料进行转录组测序，结合全基因组关联分析和RNA-seq，发现了一个谷胱甘肽-s-转移酶（GST）基因簇显著上调（BnaC06g31020D、BnaC06g31030D和BnaC06g31040D）；同时发现接种前后的差异表达基因富集到了谷胱甘肽代谢过程。另外，我们利用甘蓝型油菜全基因组重测序(5x)数据，进行GST候选基因关联分析，确定BnGSTU12 (BnaC06g31030D)为抗病候选基因。本研究拟在前期研究基础上，分析BnGSTU12基因在甘蓝型油菜菌核病抗性中的功能，初步探讨其抗病机制，并开发与菌核病抗性相关的功能标记。本研究对油菜抗病育种具有理论和实践意义。

菌核病是油菜生产中的主要病害，可造成油菜产量降低和品质下降。前期我们通过全基因组关联分析和接种核盘菌的抗感材料进行转录组测序，找到候选BnGSTU12基因。对其表达模式进行分析，发现BnGSTU12基因在叶片、根和50d种子种表达量较高，主要受激素ETH、SA、MeJA诱导表达，而在非生物胁迫PEG和NaCl后，其表达量较对照基本无变化。构建超表达载体，遗传转化拟南芥，对野生型和超表达拟南芥进行核盘菌接种，结果表明超表达拟南芥的菌斑面积显著低于对照，菌核病抗性较野生型显著提高。BnGSTU12基因主要受激素诱导表达，激素胁迫后，我们分析了超表达拟南芥和野生型的根长，发现超表达拟南芥的根长较野生型显著增加。同时，我们利用超表达载体瞬时转化烟草，接种核盘菌后进行台盘蓝染色，发现瞬时表达BnGSTU12后的烟草较对照颜色较浅，对其菌斑面积进行测定，发现瞬时表达BnGSTU12后的烟草菌斑面积显著小于对照。利用VIGUS载体转化型油菜，接种核盘菌后，发现转基因油菜中的菌核病抗性较野生型显著降低，表明BnGSTU12在油菜菌核病抗性过程中起着重要作用。另外，我们利用ZS11中111个组织器官的转录组数据，对GST基因和转录因子进行共表达分析，鉴定出BnGSTU12的上游调控候选基因WRKY33，对BnGSTU12的启动子进行分析，发现其中含有WRKY33的结合位点TTGAC和GTCAA。本研究对油菜抗病育种具有理论和实践意义。