| Phaseolus vulgaris L.is an annual herb of the genus Leguminosae,which is widely planted all over the world and has high edible value.However,P.vulgaris L.with too much fiber will affect their taste,so it is very important to cultivate low-fiber and high-quality bean varieties for the development of agriculture.In order to explore the action mechanism of cellulose synthase CesA gene in P.vulgaris L.with different fiber levels,15 PhvulCesA genes were identified by genome analysis,and their expression patterns in different tissues of two kidney P.vulgaris L.varieties ’Baizhenzhu’ and ’Yuguan’ were compared.The resistant buds of kidney bean were successfully obtained by genetic transformation of ’Baizhenzhu’ with CRISPR/Cas9 vectors,and the positive transgenic Arabidopsis thaliana plants were successfully obtained by heterologous transformation of Arabidopsis thaliana with overexpression vector.The positive transgenic Arabidopsis thaliana plants were successfully obtained by Hyg screening,PCR and RT-PCR molecular identification.The purpose of this study is to reveal the action mechanism of PhvulCesA gene in kidney bean and to provide theoretical support for cultivating bean varieties with low fiber and high quality.The results are as follows:1.A total of 15 PhvulCesA genes were identified by genome analysis,which were distributed on 7 chromosomes.Phylogenetic analysis showed that 15 PhvulCesA genes were clustered into 6 groups,and different genes in the same group had the same motif.All the 15 PhvulCES A have cellulose-synt domains,of which 11 have RING domains,13 have zf-UDP domains,and have 6 or 8 transmembrane domains.2.The results of RT-PCR and RT-qPCR analysis showed that the expression level of 15 PhvulCesA genes in bean ’Baizhenzhu’ was higher than that in ’Yuguan’.The expression of Phvul002G240200,Phvul005G010400,Phvul007G190300 and Phvul009G205200 genes was the most different between the two varieties.3.The gene editing vectors of Phvul002G240200,Phvul005G010400 and Phvul007G190300 were constructed by CRISPR/Cas9 technology.After being verified by PCR detection and sequencing,they were transferred into agrobacterium tumefaciens EHA105 to successfully infect the callus of ’Baizhenzhu’ bean and obtain resistant buds.4.The overexpression vector of Phvul007G190300 gene was constructed at the same time.After being verified by PCR detection and sequencing,it was transferred into Agrobacterium tumefaciens GV3101 and infected Col-0 Arabidopsis thaliana by flower dipping method.The detection of PCR,RT-PCR,RT-qPCR and other molecules showed that the foreign gene had been successfully introduced into Arabidopsis genomic DNA and expressed at the transcriptional level.In Arabidopsis with overexpression of bean Phvul007G190300 gene,it was observed that the root of Arabidopsis became thicker,the root hair became more and the root hair density increased significantly.Expression of Phvul007G190300 gene in transgenic Arabidopsis pods was analyzed,the results showed that Phvul007G190300 gene was expressed in the pods of three strains of Phvul007G190300-OE Arabidopsis.The cellulose content in transgenic Arabidopsis pods was determined.The results showed that the cellulose content in transgenic Arabidopsis pods overexpressing bean Phvul007G190300 gene was higher than that in wild type Arabidopsis,indicating that PhvulCesA gene plays an important role in fiber development.These results lay a foundation for further improvement of P.vulgaris L.varieties by means of molecular genetics. |
