Preliminary Studyof Elongator Subunit3 And 4 Genes On Disease Resistance To Sclerotinia Stem Rotin Brassica Napus

Brassica napus,a kind of common oil crop in the world,belongs to the genus Brassica.Rapeseed is a vital source of vegetable oil and an important source of plant protein in China,and can be used as food,bio-energy,animal feed and so on.The average planting area of rapes is around 7.14 million hectares inrecent years,accounting for over 20%of the global total rapeseed planting area.In China,the total rapeseed production in 2017 reached more than 13 million tons,accounting for about 17%of the world’s total rapeseed production,which has ranked up to the top three in the world’s total production（data from FACSTAT）.However,the sclerotinia stem rot has brought huge waste of resources and economic losses to agricultural production in recent years.The using of chemical controls to solve the sclerotinia stem rot can cause serious damage to the environment.The effective ways to solve this problem isto study the molecular the mechanism of Sclerotinia sclerotiorum resistance regulation of rapeseeds and study the cultivation of anti-sclerotinia stem rot rapeseeds.The Elongator has palyed an important role in regulating the immune process of advanced plants.In recent years,the functions of Bn ELP3 and Bn ELP4 in Arabidopsis immune process have been concerned,but the function of these genes has not been verified in Brassica napus.In this study,by using bioinformatics,molecular biology,plant tissue culture,microbiology and genetic engineering,Bn ELP3 and Bn ELP4 were cloned and Bn ELP3 and Bn ELP4 overexpression vectors were constructed.Transgenic plants were identified,the expression pattern of related disease resistance genes was analyzed andthe function of Elongator in the resistance of sclerotinia stem rot in rapeseed was verified.Studies have shown that advanced plants have to conduct a large-scale transcriptional reprogramming in vivo under environmental stress during growth.Elongator Complex is an important structure in the process of plant transcription and translation.It interacts with highly phosphorylated RNAPII to participate in plant transcription.Exactly,it plays an essential role in t RNA modifications at wobble uridinesor thiouridines at position 34 of the anticodon,which has ensured the fidelity and efficiency of translation.Disease resistance and growth-related functions of the elongator complex have been reported in strawberries,tomatoes and mostly in Arabidopsis.The response to biotic stress in the Elongator Complex subunits ELP3 and ELP4 has been reported in Arabidopsis.The two constructed vectors were transferred into Brassica napus L.by Agrobacterium,and the positively analyzed transformed plants were preliminarily identified and the expression patterns of related disease resistance genes were analyzed.The obtained results of this research are as follows:1.The Bn ELP3 and Bn ELP4 gene predicted sequences were searched in the NCBI database,and specific primers were designed according to the corresponding sequences,and the Bn ELP3 and Bn ELP4 genes were successfully cloned from Brassica napus.2.Two vectors p CAMBIA1300-2301-Bn ELP3 and p CAMBIA1300-2301-Bn ELP4 were successfully constructed.3.The above successfully constructed vector was transformed into competent Agrobacterium EHA105,introduced into the explants of Brassica napus Westar by Agrobacterium inoculation,and the resistant plants were screened and differentiated to obtain positive plants by kanamycin.After being identified by kanamycin PCR,3 strains of p35S::Bn ELP3 positive plants and 6strains of p35S::Bn ELP4 positive plants were cultured.Subsequently,6 strains of T₁generation p35S::Bn ELP3 positive plants were obtained,and 19strains of T₁ generation p35S::Bn ELP3 positive plants were obtained.3.The above successfully constructed vector was transformed into competent Agrobacterium tumefaciens EHA105,introduced into the explant of Westar using the Agrobacterium infection,and transformed plants were screened by kanamycin.After being identified by kanamycin PCR,3 p35S::Bn ELP3T₀ positive plants and 6 T₁ positive plants were obtained;6 p35S::Bn ELP4T₀ positive plants and 19 T₁ positive plants were obtained.4.Based on the expression analysis of obtained plants,it was found that the expression of3 strains in T₀ p35S::Bn ELP3 was significantly up-regulated,and the expression of 2 strains in T₀ p35S::Bn ELP4 was significantly up-regulated,and the expression of 6 strains in T₁p35S::Bn ELP3 was significant up-regulated,the expression of 17 strains in T₁p35S::Bn ELP4was significantly up-regulated.5.After in vitro inoculation of S.sclerotiorum in transgenic rapeseed leaves,the expression of related resistance genes in T₀ Bn ELP3 was analyzed.It was found that the relative expression levels of some disease resistance related genes ELP4,PDF1.2 and PR1were significantly up-regulated at 36hpi.After T₁Bn ELP4 was innocated,the relative expression levels of PDF1.2,Chitinase,NPR1,ERF1,and ORA59 were significantly up-regulated.The results have showed that Bn ELP3 and Bn ELP4 have a positive regulatory effect on the function of enhancing the resistance to the sclerotinia stem rot.6.The leaf components of T₁Bn ELP3 and Bn ELP4 were extracted and the process of inhibiting the growth of Sclerotinia sclerotiorum was simulated in vitro.It was found that protein and other secondary metabolites can effectively inhibit the growth of Sclerotinia sclerotiorum in vitro.The results of this inhibition have showed that Bn ELP3 has a positive regulatory effect on the function of enhancing the resistance to the sclerotinia stem rot.The above experimental results will provide theoretical guidance and material basis for further study of the Elongator complex subunits Bn ELP3 and Bn ELP4 in enhancing the disease resistance to sclerotinia stem rot in Brassica napus L.