Research Of Differential Expression On Copper Stress In Brassica Chinensis L.

Copper is an essential trace element for plant growth and development,but an excess of copper will cause harm to plants.In this research,seedlings were cultivated with 10 mg/L Cu2+ concentration higher than standard formula ehydroponic nutrient and copper stress response genes of Brassica chinensis L.were analyzed using the cDNA-AFLP technique.On the basis of previous studies,we’ll attempt to carry out further analysis on copper toxicity and revealing the mechanism of copper toxicity.This study will provide a theoretical basis for vegetable genetics and breeding of Brassica chinensis L..The main findings were as follows:1.Gene expression profiling in response to copper stress in Brassica chinensis L.leaves by cDNA-AFLPThe genes’ expression of Brassica chinensis L.were studied by cDNA-AFLP technique.A total of 180 differentially expressed transcript-derived fragments(TDFs)were identified by selective PCR amplification using 135 primer combinations.The TDFs included 77(42.8%)upregulated,61(33.9%)downregulated and 42(23.3%)transiently expressed genes.152 TDFs were recovered,sequenced,and 151 reliable sequences were obtained.Also 151 of the reliable TDFs sequences functions were determined through BLAST search in GenBank database.Most of the 112 TDFs genes involved in energy and metabolism,protein synthesis and ion transport,Stress response,signal transduction and regulation.Out of the 151 TDFs,112 were homolog to genes with a known function,16 were similar to genes with unknown function,and 23 had no sequence homology to GenBank entries.4 homologous genes were selected for clone the full length,analysis of the structure and function and verify the expression pattern by Real-time PCR technique.2.Cloning and expression pattern of a RING zinc finger protein gene in Brassica chinensis L.Zinc finger protein plays a key role in the regulation expression of defense and response of plant.For studies of differential gene expression,cDNA-AFLP was applied in Brassica chinensis L.under copper-stress and isolated the TDF of RING zinc-finger gene.The 855bp full length cDNA of RING zinc-finger gene was isolated by rapid amplification of cDNA ends(RACE),including a 606 bp open reading frame encoding 202 amino acid residues.The molecular weight of deduced protein was 21.32 kDa with a theoretical pI of 6.87.The protein has a C3HC4-type RING finger domain.Phylogenetic analysis showed that RING zinc-finger gene had 98%similarity with rapa and blonged to C3HC4-type Zinc-finger subfamily.Real-time PCR revealed that the gene was down-regulated expression in copper stress,suggesting that it might involve in copper stress.The results would be useful for the study of functions and mechanism to copper stress responses of Brassica chinensis L.3.Cloning and expression pattern of ubiquitin-conjugating enzyme E2 gene BcUBCE2 in Brassica chinensis L.Our objective was to illustrate the response of ubiquitin-conjugating enzyme E2 gene to copper stress in Brassica chinensis L..This study designed the primers according to the TDF fragment which associated with ubiquitin-conjugating enzyme E2 by cDNA-AFLP technique and designated as BcUBCE2.The 830bp full length cDNA of BcUBCE2 gene was obtained by RACE technique.This gene included a 459bp open reading frame encoding 152 amino acids.Structural analysis revealed that the sequence contains an E2 ubiquitin-conjugating enzyme active sites and a highly conserved cysteine.Phylogenetic analysis showed that the deduced amino acid sequence of the gene was closest to Arabidopsis.RT-PCR results showed that the expression level of Bc UBCE2 gene reached the highest until stress 10d.This showed that the BcUBCE2 gene may play an important role in the response to copper stress.4.Cloning of AP2/ERF transcription factors in Brassica chinensis L.Studies had showe that AP2/ERF transcription factors participated in copper stress response.This study designed the primers according to the TDF fragment which associated with AP2/ERF transcription factor gene by cDNA-AFLP technique and obtained the 868bp full length cDNA of AP2/ERF transcription factor gene by RACE technique.This gene included a 606 bp open reading frame encoding 202 amino acid residues.The molecular weight of deduced protein was 22.88 kDa with a theoretical pI of 5.87.The protein has a transcription activation domain in carboxyl terminal and a nuclear localization signal in amino terminal,and a AP2/ERF domain including two function units WLG and YRG.Phylogenetic analysis showed that AP2/ERF transcription factor gene had 98%、98%、99%similarity with brassica napus,brassica oleracea and Sisymbrium irio and was blong to ERF subfamily.cDNA-AFLP revealed that the gene was up-regulated expression in copper stress,suggesting that it might involve in copper stresses.5.Cloning and expression pattern of chloride ion channel protein gene CIC-d in Brassica chinensis L.Chloride channel CIC-d gene plays an important role in plant defense.The primers were designed according to the TDF which associated with chloride ion channel protein gene CIC-d by cDNA-AFLP technique.The 2 752 bp full length cDNA of chloride ion channel protein gene CIC-d was isolated by RACE technique,including a 2 376 bp open reading frame encoding 792 amino acid residues.The molecular weight of deduced protein was 87.06 kDa with a theoretical pI of 8.35.The protein has a voltage-gated chlorine ion channel and two cystathionine-beta-synthase,and blonged to CIC subfamily.Phylogenetic analysis showed that chloride ion channel protein gene CIC-d had 100%similarity with Arabidopsis thaliana.Real-time PCR revealed that the expression of chloride ion channel protein gene CIC-d was increased and reached the highest until 10 days under copper stress,suggesting that it might response to copper stresses.The results would be useful to the study of its functions and mechanism in copper stress responses of Brassica chinensis L.