Journal of plant physiology(2007) 大豆中 6 个 NAC...

Journal of plant physiology(2007)

大豆中 6 个 NAC基因的克隆、序列分析和组织特异表达Molecular cloning, sequence characterization and tissue-

specific expression of six NAC-like genes in soybean(Qingchang Meng National Center for Soybean Improvement)

汇 报 人：王爽 指导教师：寇晓虹副教授

学术报告—文献阅读汇报

Contents

Introduction1

Materials and methods2

Results3

Discussion4

Gene expression and regulation is mediated by TF

Conserved N-terminus(ABCDE) and diverged C-terminus

Play role in plant development and abiotic stress

Soybean development

Cloning and sequence characterizationgenes expression analysis

Introdution

Field grown soybean

Tissue-specific Seeds development(4weeks) leaves stems roots 15 20 (DAF) floral buds 25 30 young pods 35 40 20DAF seeds 45 50

Isolate and purify total RNA cDNA lsolate DNA from yong leaflets

Materials

Select 10 NAC protein sequences of Arabidopsis

tBlastn search in soybean EST database(E 1X10-5)

Resulted genes for similarity searches

remove low-quality sequences manually Assemble into contigs with Lasergene Seqman

Design gene-specific primers for cDNA and DNA

Methods—sequence retrieval

GmNAC1-6

cDNA:RT-PCR products sequencing

Confirm identities—BLASTn in non-redundant database

DNA:PCR products sequencing

Sequence analysis of GmNAC proteins

Phylogenetic analysis

Methods—cloning and sequencing

Tissue-specific expression

Expression of developing seeds in different stages

Deposite GmNAC sequence data

Methods—expression

ATAF1 ATAF2 CUC1 CUC2 CUC3TIP AtNAC072 AtNAM NAP AtNAC

GmNAC1-6 cloning and sequencing Length 1.0 to 1.1kb a complete ORF

GmNAC1-6 Blastn in Genebank GmNAC5 correspond to AF532619

Other novel genes

Results—isolation

DNA :PCR products sequencing1.4 to 2.0kb 3 exons and 2 introns

Introns:conservative GT-AG junctions Exons: the first two—conserved N- the last one—divergent C- Molecular modeling: protein—α β

Results—genomic structure

Deduced proteins

Results—amino acid sequence analysis

Results—sequence similarities analysis

a:Number of amino acidsb:nucleotide acid identitiesc:overall amino acid/NAC domain

Results—tissue-specific expression

1.leaves 2.young stems 3.roots 4.floral bus and flowers5.young pods 6.developing seeds 20DAF

Results—different stages seeds expression

15 20 25 30 35 40 45 50 DAF

Results—different stages seeds expression

GmNAC1:NAPGmNAC2:ATAFGmNAC3/4:AtNAC3GmNAC5:NAMGmNAC6:TERN

Discussion

Seed filling Seed development 4-5weeks

15 to 45 DAFCell division and expansionSynthesize storage matters

Similar expression Similar regulatory network

GmNAC1/5 may be involved in diverse process

实验计划同源序列查找

Sol genomicis network

多序列比对，构建进化树ClustalX MEGA(NJ/MP)

目的基因结构和编码蛋白序列分析内含子和外显子结构

蛋白理化性质 Protparma 、氨基酸疏水性图谱 ProtScale结构域 Pfam 和 SMART 、信号肽 SignalP

跨膜区域 TMHMM 、 DAS 、 PHDhtm 亚细胞定位 TargetP 、 PSORT 、 SubLoc 、 BaCelLo

实验计划

二级结构分析 SOPMA 、三级结构建模 SwissModel

数字表达分析TED digital expression

目的基因特异表达分析Field grown Tomato

Tissue-specific Fruit development