| As the first contact surface between plants and external environment,waxy is sensitive to environmental action and can resist external stress by regulating its own physiological mechanism,including biological stress and abiotic stress,which has important biological function.As a widely cultivated plant resource of Tibetan Plateau,Turnip(Brassica rapa var.rapa)has a powder / powder-free phenotype,and the powder phenotype is more drought-resistant than the powder-free phenotype.To analyze candidate genes controlling waxy traits,turnips KTRG-B48A(powdery phenotypes)and KTRG-B48B(powdery phenotypes)were used to screen candidate genes regulating waxy phenotypes of turnips by BSA sequencing.The transgenic function was verified to analyze the difference of candidate genes in the regulation of Turnip varieties.The main results are as follows:(1)Mapping of the regulating wax genes in turnipThis study used BSA to locate the waxy gene of turnip,and to construct F2 isolated population by using wax phenotype(KTRG-B48A)and wax-loss phenotype(KTRG-B48B)as parents.After data quality control,comparison with turnip genome and SNP-Indel analysis of differential loci,the candidate regions of waxy gene on chromosome 3 were obtained.According to candidate gene annotation and expression results,the waxy phenotype of turnip was controlled by CER3 gene.(2)Cloning and functional analysis of candidate gene CER3 for turnipCER3 is a key gene in plant waxy synthesis.In order to analyze the differential regulation of CER3 in different waxy varieties of turnip,its full length was cloned and its function was analyzed.Through the protein sequence alignment of turnip CER3 gene between varieties,it was found that the point mutation at the 345 th locus caused the deletion of a spiral domain,which may promote the wax synthesis of CER3 gene and lead to the change of wax between varieties.The CER3 function was verified by genetic transformation of wild-type arabidopsis thaliana.The growth of waxy BrrCER3-48 A lines of turnip transgenic arabidopsis thaliana under drought stress was significantly better than that of wild-type and waxy BrrCER3-48 B lines,with higher root length,fresh weight and survival rate,indicating that waxy transgenic lines were resistant to drought stress.(3)Effects of drought treatment on genes related to waxy anabolic pathwayThe expression of all related genes in the whole metabolic pathway was analyzed in combination with turnip transcriptome data.The expression of KCS、CER1、CER3、CER10 and MAH1 waxy related genes in waxy KTRG-B48 A turnips was higher than that in waxy KTRG-B48 B,and drought stress significantly induced the up-regulation of BrrKCS1 and BrrKCS2.KCS plays an important role in the synthesis and metabolism of wax,so the gene family in turnip is identified and expressed.Twenty-eight BrrKCS genes were identified for the first time genome,and phylogeny showed that the gene family KCS was divided into eight subfamilies in the turnip.The different expression levels of turnip BrrKCS genes in different developmental stages and drought conditions indicate that the BrrKCS gene family has undergone functional differentiation during long-term evolution,and it may play an important role in promoting the waxy synthesis of turnip and enhancing the tolerance of turnip to drought stress.Our analysis provides an important research basis for understanding BrrKCS potential role in regulating turnip waxy synthesis and response to drought stress. |
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