| Nucleotide binding site(NBS)is a class of disease resistance genes that play an important role in plant disease resistance.At present,NBS gene has been found in Tomato,Arabidopsis,Potato and many other plants,but there are few studies on the function of strawberry NBS gene.In this study,the coding region of FaNBS13 gene was cloned from the ’Yanli’ strawberry in octaploid cultivation,its over-expression vector and silencing vector were constructed and transgenic strawberry plants were obtained.The resistance of transgenic strawberry plants to FaNBS13 gene was proved by inoculation with Botrytis cinerea in order to lay an important foundation for revealing the function of strawberry overexpression gene.The main results are as follows:(1)The FaNBS13 gene was cloned from ’Yanli’ strawberry by RT-PCR technique.The full-length coding region of the FaNBS13 gene was 1776 bp,encoding 592 amino acids.using DNAMAN 6.0 software,the protein structure was analyzed and the main core domain was found to be LRR.Subcellular localization assay showed that FaNBS13 protein was mainly located in the nucleus.(2)qRT-PCR method was used to detect the relative expression of FaNBS13 gene in different tissues and organs of ’Yanli’ strawberry.It was found that FaNBS13 gene was mainly expressed in leaves,and the expression level of FaNBS13 gene in leaves was 2.3 times higher than that in roots.With the increase of leaf senescence expression;The expression levels in sepals and stamens were 1.2 and 1.9 times,respectively,and changed significantly with the ripening of fruits.(3)Overexpression vectors and silencing vectors of FaNBS13 gene were constructed using pRI101-AN and pRI101-RNAi vectors,and transformed into strawberries by Agrobacterium-mediated genetic transformation to obtain transgenic strawberry lines 10 overexpressing FaNBS13 gene.Six transgenic strawberry lines that silenced the NBS13 gene.Agrobacterium-mediated injection of the bacterial liquid into the ’Yanli’ strawberry fruit,and the Botrytis cinerea pathogen was inoculated after the fruit matured.The observation of the pathogenesis and gene expression analysis showed that the FaNBS13 gene could improve the Botrytis cinerea resistance of the strawberry fruit.(4)The ’Ruegen’ strawberry tissue culture seedlings were treated with IAA,SA,MeJA and ABA,respectively,and samples were taken at 0 h,12 h,24 h,48 h,72 h,96 h,and detected by qRT-PCR.The expression level of NBS13 gene was found to be up-regulated under the treatment of four hormones,and the expression level of NBS13 was relatively high under IAA treatment.(6)Select Japanese strawberry varieties ’Sachinoka’ and ’Tochiotome’ with the same growth status,European and American strawberry varieties ’Sweet Charlie’ and ’Tudla’ and new varieties of self-bred strawberries ’Yanli’ leaves,the expression of NBS13 gene was detected by inoculating Botrytis cinerea,and the expression level of NBS13 gene was up-regulated compared with uninoculated leaves,and the expression of NBS13 gene in Japanese varieties after infection The increase is more than that of European and American varieties. |
