journal of plant physiology(2007) 大豆中 6 个 nac...
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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
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