Cloning And Function Analysis Of Chinese Cabbage BrANT Gene(Brassica Rapa L.ssp. Pekinensis)

Chinese cabbage(Brassica rapa L. ssp. pekinensis) is a widely cultivated and economically important vegetable crop in Asia. Chinese cabbage originated in China, and has now become increasingly popular in other countries. Chinese cabbage has a tight leafy head, which is a storage organ for the cabbage and is the part that is commonly eaten. The size of leafy head is an important economical trait, and also a commodity trait. Therefore, how to understand the molecular mechanism of leafy head size development has always been the key question of the genetic improvement in Chinese cabbage. However, up to date, little is known about the molecular regulation of leafy head size development. Recently, it is found that ANT genes, belonging to AP2 subfamily transcription factors, are involved in the control of leaf organ size and shape in Arabidopsis. In this study, base on the complete genome sequence data of Chinese cabbage line Chiifu-401, the genome-wide analysis of AP2 gene subfamily in Chinese cabbage was carried out, including members of Br ANT protein family, and their structures and expression patterns. Moreover, the high-throughput RNA sequencing(RNA-Seq) was also employed to investigate the different expression pattern between transgenic Arabidopsis plant(35S:Br ANT2) and its wild type, because the regulation of leaf size is very complex, and related to many other functional genes and transcript factors. The results are as follows.1. Bioinformatic analysis of AP2 protein family in Chinese cabbageBased on the recently sequenced Chiifu genome and annotated genes, we found that there were 24 Br AP2 genes in Chinese cabbage, which unevenly distributed in ten chromosomes of Chinese cabbge. The numbers of introns for Br AP2 genes were different, most of which had 6 introns. The analysis on the sequence of Br AP2 proteins indicated that these proteins contained two conserved domains(R1 and R2 domain). According to the number of amino acid residues in R1 and R2, the Br AP2 subfamily was divided into seven types, of which ‘73, 65‘, ‘70, 65‘ and ‘63, 64‘ were three main types. The phylogenetic tree of Arabidopsis, Chinese cabbage and rice AP2 subfamily were constructed by MEGA4.1 through Bootstrap-neighbour joining method. These three plant AP2 genes were classified into eight subgroups, and Br ANT were located in groupⅥ. Based on the phylogenetic tree, we found that Br ANT proteins were closed to the At ANT proteins, but remoted to the Os ANT proteins.2. Expression patterns of Br ANT genesWe used RT-PCR essay to examine the expression pattern of all Br ANT genes in different tissues, including root, dwarf stem, old leaf, young leaf, flower and flower bud, and found that most of Br ANT genes had various expression levels in these tissues. In addition, the different expression level of Br ANT genes under NAA treatment for different time was detected, in which Br ANTs were up-regulated in early time and then tend to balance. Further analysis indicated that the Br ANT genes containing the same domain had similar expression patterns. These results suggested that these genes could play different roles in regulating leaf size development of Chinese cabbage.3. Cloning and function analysis of Chinese cabbage Br ANT2 geneTo understand the function of Br ANT genes, the BrNAT2 gene was selected as an example for further analysis. The Br ANT2 gene, whose full-length c DNA consists of 2250 nucleotides with a 1671-nucleotide coding sequence, was located on chromosome 03. The coding sequence of Br ANT2 was predicted to encode a 556-amino acid polypeptide.The expression pattern of Br NAT2 gene in various inbred lines with different size of heading leaves was analyzed by q RT-PCR essay. The results indicated that the expression pattern of Br ANT2 gene was positive correlated to the size of heading leaves among these inbred lines. In addition, the over-expression of Br ANT2 gene could increase the size of Arabidopsis transgenic plant organs, as well as the one thousand seed weight.4. Transcriptome analysis of transgenic Arabidopsis plant(35S:Br ANT2) and its wild typeThe transcriptome analysis for leaves of transgenic Arabidopsis plant(35S:Br ANT2) and its wild type grown 30 days was carried out using high-throughput RNA sequencing. Comparative transcriptome analysis demonstrated that 119 genes exhibited differential expression patterns, including 40 up-regulated genes and 79 down-regulated genes in transgenic plant. We also found that Br ANT genes regulated the size of plant leaves through a complex gene network, in which Br ANT genes could up-regulate many related genes including cell cycle and dividing regulatory factor, calmodulin and photomorphogenesis related genes, and down-regulated extension protein coding gene, senility related gene and so on, to regulate the size of plant leave.