Fine Mapping And Expression Characteristics Of The Etiolated Mutant Gene Brpem1 In Chinese Cabbage

Leaf color mutation is a general phenomenon in nature.A variety of leaf color mutants have been created and identified in many crops using different methods.The main phenotype of the leaf color mutant is the etiolated mutation,which has a lot of research on the mutant phenotype,physiological mechanism,genetic characteristics and mutant genes.Most etiolated mutants have decreased photosynthetic pigment content,photosynthetic rate and may even lead to plants death.Chinese cabbage(Brassica campestris ssp.pekinensis)is a biennial vegetable come from China belonging to the Brassica.It is a vegetable crop commonly cultivated in China.Since the release of the Chinese cabbage genome sequencing result in 2011,the genome research of Chinese cabbage has entered the functional genomic era.It is an important method of functional genomics to analyze and elucidate the function of genes by artificial mutagenesis.A stable inherited Chinese cabbage etiolated mutant pem1(plants etiolation mutant 1)was obtained by the combination of 60Co-? ray treatment and free microspores cultured.Based on the analysis of the physiological characteristics and genetic characteristics of mutant pem1,this study performed fine mapping of mutant gene and study of candidate gene function.The main findings are as follows.1.Morphological characteristics,photosynthetic characteristics and chloroplast ultrastructure of pem1During the whole growth period of the pem1,the plant showed etiolated and the leaf head was small.Compared with wild-type 'FT',the content of chlorophyll a,chlorophyll b,total chlorophyll and carotenoid were significantly decreased in the leaves of pem1,the photosynthetic rate decreased and chloroplast fluorescence kinetic parameters were significantly decreased,indicating that the decrease in pigment content affects the photosynthesis of pem1.The chloroplasts of the pem1 were degraded,the thylakoids grana stacking were sparsely,the chloroplasts were irregular in shape and there were distinct starch granules.2.Fine mapping of mutant gene Brpem1Analysis of the genetic characteristics of the pem1 showed that the mutant trait was controlled by a pair of recessive nuclear genes.The SSR marker was used to map the mutant gene Brpem1 between SSRlx-49 and SSRlx-69 on chromosome A03,and the genetic distances from the mutant gene were 0.32 c M and 0.17 c M,respectively.Comparing the Chinese cabbage genomic sequence information showed that the mapping region was about25.88 kb,including seven genes.By cloning and sequencing of these seven genes,it was found that only the promoter region of the gene Bra024218 has a 30 bp deletion compared with wild-type 'FT'.The homologous gene of Bra024218 was AT4G28210 in Arabidopsis.Its function was embryo defect(EMBRYO DEFECTIVE 1923,EMB1923)and influenced thechloroplast development,so Bra024218 was predicted to be the candidate gene for Brpem1.3.Analysis the expression patterns of candidate gene Bra024218Analysis of sequence variation in mutant gene showed that the promoter region of the gene Bra024218 has a 30 bp deletion in the pem1 compared with the wild type 'FT'.The deletion fragment has a promoter core element CAAT box and the deletion region may be the core region of the promoter.The results of q RT-PCR analysis indicated that the candidate gene Bra024218 was expressed in root,stem,leaf,bud,flower and seed pod,but the expression in pem1 was significantly weakened.The expression levels of the candidate gene Bra024218 in the different development stage leaves showed that the expression of the pem1 was significantly reduced.GUS analysis indicated that the candidate gene Bra024218 was expressed in almost all tissues,but the expression was relatively weak in the pem1,and the GUS enzyme activity was also significantly decreased.The results of subcellular localization indicated that the candidate gene was only expressed in the chloroplast.4.The reason for chlorophyll deficiency in pem1A comparative transcriptome analysis of wild type 'FT' and the pem1 showed that many of the differentially expressed genes were associated with leaf color mutations.Among the photosyntheic system I and photosyntheic system II related genes,many genes were significantly down-regulated in the pem1.The content of chlorophyll biosynthesis precursors and related gene expression patterns were analyzed and compared.The results showed that the content of chlorophyll biosynthesis precursors was decreased and the content of some key precursors also decreased significantly.The expression levels of some genes encoding key enzymes in the chlorophyll biosynthesis were also significantly decreased.Among the differentially expressed genes,the expression levels of many genes encoding Fts Hi protein were significantly up-regulated,which may affect the chloroplast development of pem1.The expression levels of the genes encoding the chlorophyll a-b binding proteins were significantly down-regulated,which may affect the light-harvesting ability of the pem1.