Studies On The Function Of Tomato Annexin SlAnn5 In Resistance To Bemisia Tabaci

Bemisia tabaci,the most destructive and difficult-to-manage omnivorous pest worldwide,cause great damage to agricultural production.Spraying insecticides is the most common method in present agriculture cultivation.The extensive use of insecticides will not only lead to drug resistance of B.tabaci,but also cause other problems such as food safety.Therefore,it is of great significance to find the B.tabaci resistant genes in plants and cultivate B.tabaci resistant varieties to reduce the harm of B.tabaci.Plant annexin is widely involved in the regulation of plant growth and response to environmental stress.In our previous study,we found that the pepper CaAnn9 gene positive regulatory resistance of pepper to B.tabaci directly or indirectly.It is reasonable to speculate that the tomato annexin SlAnn5,the homologous of the pepper CaAnn9 gene,might also involved in the resistance response to B.tabaci in tomato.In this paper,the roles of in in tomato resistance to B.tabaci were researched.To achieve this goal,the tomato annexin family information were identified by bioinformatic technique and the characteristics of SlAnn5 protein were analyzed.Using Agrobacterium-mediated genetic transformation technology,SlAnn5 homologous or heterologous transgenic tomato and Arabidopsis materials were obtained,and the effect and mechanism of tomato SlAnn5 against B.tabaci in tomato and Arabidopsis were preliminary studied.The main findings were as follows.1.Bioinformatics analysis showed that the amino acid sequences of the 10 SlAnns in the tomato annexin gene family were highly conservative,possessing typical structural features of plant annexins,including three to four annexin repeat domains,a type II calcium binding site,and an IRI motif capable of binding to F-actin.Phylogenetic tree analysis showed that SlAnn5 was on the same evolutionary branch with AtAnn4 in Arabidopsis and CaAnn9 in Pepper.The results of subcellular localization experiments showed that SlAnn5 might be located in the cell membrane and cytoplasm,but not in nucleus.Quantitative fluorescence level analysis showed that the expression level of SlAnn5 was significantly increased under the induction of Bemisia tabaci feeding.2.Through transgenic technology,the SlAnn5 overexpressed tomato material and heterologous overexpressed Arabidopsis were constructed.The results of insect resistance experiments showed that,compared with wild-type material,the selective rate of B.tabaci for the overexpressed plants were significant reduced.The egg laying number of Bemisia tabaci on the overexpressed material were reduced significantly.The insect population rate was decline,and the hypersensitive reaction was weakened after six days of B.tabaci feeding.The above results indicates that the SlAnn5 gene was involved in the process of resisting Bemisia tabaci in tomato.The insect resistance experiments were also performed in Arabidopsis annexin AtAnn4 T-DNA insert mutant anann4 and the AtAnn4 promoter complementary plants Proatann4:AtAnn4.The results also confirmed the resistant effect of AtAnn4 to B.tabaci.3.In order to reveal the resistance mechanism of SlAnn5 gene to B.tabaci,the plants overexpressing tomato SlAnn5 were used as materials,and treated them with B.tabaci.The change of antioxidant activity,the JA and SA contants and the resistance-related gene expression levels were determined.The results showed that after B.tabaci infection,the antioxidant enzyme activity of wild-type tomato in the control group was decreased significantly,while the enzyme activity of SlAnn5 gene overexpressing strain was not changed significantly,which indicated that the SlAnn5 might regulated the resistance by regulated the ROS balance in tomato.Further research revealed that the abundant expression of SlAnn5 increased the content of JA but decreased the content of SA in the leaves of tomato.Results from qRT-PCR indicated that the overexpression of SlAnn5 induced the significant increase in the expression levels of JA biosynthesis-related genes such as SlAOC and SlOPR3,and decreased expression levels of SA transport and signal transduction-related genes such as SIEDS5 and SIPAD4.The above results suggested that SlAnn5 gene was involved in the tomato resistance to Bemisia tabaci,and it might improve the tomato resistance to Bemisia tabaci by participating in ROS pathway and the pathway related to JA synthesis.4.Using SlAnn5 as the bait to screen the tomato cDNA yeast library induced by Bemisia tabaci feeding,15 protein that might interact with SlAnn5 were initially screened.The results of comparative analysis showed that the screened protein types included transcription factors,kinases,proteins related to nitrogen metabolism regulation,and stressresponsive proteins.Among the screened protein,two rubp carboxylated oxygenases,RBCS2A and RBCS-3B,had the highest recurrence rate.