甜菜夜蛾取食后雪里蕻硫苷变化和差异表达基因的研究

发布时间：2018-09-08 21:36

【摘要】：硫代葡萄糖苷(简称硫苷)是十字花科植物中典型的防御代谢产物。在转运蛋白的作用下,植物体内的硫苷形成了非均匀的分布模式,易受昆虫取食的部位容易积累更高浓度的硫苷。芥菜中含有的特异硫苷组分是其表现出抗虫性状的主要原因,已有研究证实,芥菜中主要硫苷的降解产物对幼虫具有致死作用。然而,芥菜中硫苷的分布模式及硫苷介导的防御分子机制尚未报道。所以本试验中,我们检测了叶用芥菜雪里蕻不同组织、不同生长时期和不同叶位的硫苷组分及含量,讨论了芥菜中硫苷分布的生理和生态意义。此外,还研究了芥菜中硫苷介导的响应杂食性害虫甜菜夜蛾取食的分子防御机制。得到如下主要研究结果:(1)芥菜的根和叶中硫苷组分和含量存在较大差异。在子叶期向十六叶期生长发育的过程中,叶中的总硫苷含量逐渐减少,但八叶期之后总硫苷含量无显著差异,且二丙烯基硫苷是叶中主要的硫苷。相反,芥菜根中的总硫苷含量在子叶期向十六叶期生长发育中呈上升趋势。我们在根中还检测到了叶中未检测到的2-苯乙基硫苷,且该硫苷在八叶期成为根中含量最高的硫苷,而十二叶期和十六叶期根中二丙烯基硫苷则是含量最高的硫苷。(2)对九叶期芥菜的不同叶位之间硫苷含量进行分析,结果表明,越幼嫩的新叶硫苷含量越高。(3)芥菜总硫苷在甜菜夜蛾取食1h后没有明显变化,但在取食24h后显著增加(24.7%)(P0.05)。甜菜夜蛾取食24h后脂肪族和吲哚族硫苷分别增加了22.1%和239.4%。二丙烯基硫苷在取食24h后增加了24.0%(P0.05),其含量约占总硫苷的96.0%。3-丁烯基硫苷也在芥菜被取食后显著增加(P0.05)。除了4OHI3M以外,所有的吲哚硫苷都在取食1h后就显著增加,I3M增加最多。而在甜菜夜蛾取食24h后所有的吲哚族硫苷含量都显著增加。(4)转录组测序分析显示,三个样本(CK,1 h和24 h)分别获得20.7,20.6和20.0百万个clean reads。测序片段组装后共获得60,948个独立基因,基于七个数据库的相似性分析共得到40,580个功能注释独立基因。芥菜被取食后,硫苷合成、降解和转运的53个同源基因的表达量几乎全部上调。同时,植物激素JA,ET和SA基因的表达量也呈上升趋势。JA基因的表达量在取食1小时后显著上升,不管是取食1h还是24h后,JA基因的表达量上升幅度明显都高于ET和SA基因。我们的试验结果为进一步研究硫苷介导的抗虫防御机制和芥菜中硫苷代谢关键基因的功能奠定了基础。
[Abstract]:Glucosinolate (glucosinolate) is a typical defense metabolite in cruciferous plants. Under the action of transporter, the glucosinolates in plants form a non-uniform distribution pattern, which is easy to accumulate a higher concentration of glucosinolate in the parts vulnerable to insect feeding. The specific glucosinolates contained in mustard are the main reasons for their insect-resistance. It has been proved that the degradation products of main glucosinolates in mustard have lethal effect on larvae. However, the distribution pattern of glucosinolate in mustard and the defense molecular mechanism mediated by glucosinolate have not been reported. Therefore, in this experiment, we detected the composition and content of glucosinolate in different tissues, different growth stages and different leaf positions, and discussed the physiological and ecological significance of the distribution of glucosinolate in Brassica juncea. In addition, the molecular defense mechanism of glucosinolate mediated response to the feeding of beet Spodoptera exigua in mustard was studied. The main results were as follows: (1) there were great differences in the composition and content of glucosinolate between the roots and leaves of mustard. During the growth and development from cotyledon stage to hexadecap stage, the total glucosinolate content in leaves decreased gradually, but there was no significant difference in total glucosinolate content after eight leaf stages, and diallyl glucosine was the main glucosine in leaves. On the contrary, the total glucosinolate content in mustard root increased from cotyledon stage to hexadecyl stage. We also detected 2-phenylethyl glucosinolate, which was not detected in the leaves, and it became the highest level of glucosinolate in the root at the eight-leaf stage. However, diallyl glucosinolate was the highest content in the roots of mustard at 12 and 16 leaf stages. (2) the contents of glucosinolate in different leaf positions of mustard at nine leaf stage were analyzed, and the results showed that, The content of glucosinolate in the young leaves was higher. (3) the total glucosinolates of mustard did not change after feeding for 1 hour, but increased significantly (24.7%) after 24 hours (P0.05). After feeding for 24 hours, the glucosinolates of aliphatic group and indole group increased by 22.1% and 239.4%, respectively. The content of diallyl glucosinolate increased by 24.0% (P0.05) after ingested for 24 hours, and the content of diallyl glucosinolate increased significantly (P0.05) after ingesting mustard (P 0.05), which accounted for 96.0.3-butenyl glucosinolates of total glucosinolate (P < 0.05). Except for 4OHI3M, all indolethioside increased significantly after feeding for 1 hour. However, after 24 hours of feeding, all indolyl glucosinolates were significantly increased. (4) the three samples (CK,1 h and 24 h) obtained 20.7G 20.6 and 20.0 million clean reads., respectively. (4) the transcriptome sequencing analysis showed that the three samples (CK,1 h and 24 h) were 20.6 and 20.0 million clean reads., respectively. A total of 60948 independent genes were obtained after the sequencing fragments were assembled, and 40580 functional annotated independent genes were obtained based on similarity analysis of seven databases. After the mustard was fed, the expression of 53 homologous genes that were synthesized, degraded and transported were almost upregulated. At the same time, the expression of plant hormone JA,ET and SA genes also showed an increasing trend. The JA gene expression increased significantly after 1 hour of feeding, and the expression of JA gene was significantly higher than that of ET and SA genes after 1 hour or 24 hours of feeding. Our results provide a basis for further study of glucosinolate mediated anti-insect defense mechanism and the function of key genes involved in glucosinolate metabolism in mustard.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S436.344

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