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cDNA MICROARRAY MONITORING OF mRNA EXPRESSION PATTERN IN DIFFERENT RICE TISSUES AND STEM TISSUE INDUCED BY PHYTOHORMONE ABSCISIC ACID

作　者: Daniel Cabezas Montero.
导　师: Dong Haitao；Li Debao
学　校: 浙江大学
专　业: Philosophy in Plant Pathology
关键词: cDNA microarray tissue-specific expression intron repeated gene Abscisic acid (ABA) cytochrome C
分类号: S511.03
类　型: 硕士论文
年　份: 2001年
下　载: 25次
引　用: 0次
阅　读: 论文下载

内容摘要

cDNA microarray consisting of 1330 unigenes derived from rice endosperm cDNA library was applied to monitor the transcript abundance of different tissues ( root, stem and leaf) in rice line Zhongzao 21.The results demonstrated that microarray could be used to monitor the expression levels of thousands of gene in different tissues simultaneously.By analyzing the results of hybridization we got one tissue-specific full-length cDNA fragment (H012d03), which had none, trace and intensive expression level in tissues of root, stem and leaf respectively. Northern blotting confirmed the results.Electronic PCR results suggested that :his gene lie in the structural gene of 23S rRNA belong to rice chloroplast genome. No similar rice EST had been found in the public database.Some research has proved that there was one intron repeated gene encoding I-CPAI protein in 23S rRNA of chloroplast genome of green alga. The gene can transcript into mRNA with poly A tail and encode protein with bioactivity. In rice, we found it is the first time in the world that in the structural gene of 23S rRNA of chloroplast genome, there also have the intron repeated gene could transcript into mRNA with poly A tail.Meanwhile, Another cDNA microarray with 4370 unigenes from library of rice endosperm and leaf tissue was also used to detect the expression level of mRNA from stem tissue of rice treated by water and plant hormone ABA. Results showed that the expression levels of 5 genes were depressed by phytohormone ABA. Reverse Northern blotting confirmed that one of the 5 genes (H024g06) was really depressed by ABA. Bioinformatics analysis showed that the gene H024g06 is the same ascytochrome C gene. Previous researches revealed that cytochrome C gene was associated with such stress response as drought and coldness, while ABA could induce the stress response of plant. Therefore our results suggested that the gene cytochrome C may have certain function of mediate during the stress response induced by phytohormone ABA.

全文目录

Content  6-8
英文摘要  8-10
1． Introduction  10-11
2． Literature Review  11-20
  2．1． Microarray  11-14
    2．1．1 Developing  11-12
    2．1．2 Application  12
    2．1．3 Advantages and disadvantages  12-13
    2．1．4 Perspective  13-14
  2．2． General aspects about Abscisic acid （ABA）  14-20
    2．2．1 Abscisic acid functions and mechanisms  14-16
    2．2．2 Abscisic acid signal transduction research  16-17
    2．2．3 Abscisic acid in environmental stress condition  17-19
    2．2．4 Perspective  19-20
3． Materials and Methods  20-32
  3．1． Preparation of rice samples  20
    3．1．1 ABA treatment and sampling of three rice lines  20
    3．1．2 Seedling growing of Zhongzao 21 andsampling of different tissue at different development stage  20
  3．2 Total RNA isolation  20-21
  3．3 RNA electrophoresis analysis  21
  3．4 Isolation of Poly A mRNA from total RNA  21-22
  3．5． Preparation of total cDNA probes  22
  3．6 cDNA Microarray preparation  22-23
    3．6．1． cDNA library plating  22-23
    3．6．2． Clones Inoculation  23
    3．6．3． Plasmid Store （LB/glycerol 1:1）  23
  3．7． Plasmid extraction by Minipreparation using MultiScreen 96-Well Filter plates  23-26
    3．7．1． Sequencing and analysis  25
    3．7．2． Polymerase Chain Reaction （PCR）  25
    3．7．3． PCR product cleaning  25
    3．7．4． Database building  25-26
    3．7．5． Templete Dilution and Polymerase Chain Reaction （PCR）  26
    3．7．6． PCR product precipitation  26
    3．7．7． PCR product denaturation  26
  3．8． Preparation of Array  26-27
  3．9 Chip hybridization  27-29
    3．9．1． Prehybridization  27-28
    3．9．2． Hybridization  28
    3．9．3． Membranes washing  28-29
    3．9．4． X film autoradioactivity exposure  29
    3．9．5． X-film Analysis and differential display gene search  29
  3．10． Reverse Northern．  29-30
  3．11 Northern blotting  30-31
  3．12 Full-length sequence and bioinformatics analysis  31-32
4． Results and Analysis．  32-53
  4．1． cDNA microarray analysis of tissues-specific genes in rice  32-42
    4．1．1 Preparation of PCR products of 1330 unigenes and micro-arraying process  32-33
    4．1．2 Isolation of total RNA from rice different tissues of root, stem and leaf, and synthesis of cDNA first-chain probes  33-34
    4．1．3 Identification of differentially expressed genes in rice different tissues （root, stem and leaf）  34-35
    4．1．4 Northern blotting: to test the expression levelof H012d03 clone in different tissues of rice  35-36
    4．1．5 DNA full sequence analysis of H012d03 clone  36
    4．1．6 the electronic PCR location and bioinformatics analysis of H012d03 clone  36-42
  4．2 cDNA microarray detection of mRNA expression of 4370 genes in rice induced by phytohormone ABA．  42-53
    4．2．1 cDNA chip preparation  42
    4．2．2 Isolation of total RNA from rice stem treated with ABA and the synthesis of cDNA probes  42-44
    4．2．3 The bioassay of fluctuation of gene expression in rice stem tissue induced by phytohormone ABA．  44-45
    4．2．4 Data of a number of positive spots obtained through chip hybridization  45
    4．2．5 Using reverse northern blot to confirm the expression situation of the 5 cDNA clones inhibited by ABA treatment  45-46
    4．2．6 Bioinformatics analysis of Cytochrome C clone．  46-53
5． Discussion  53-55
  5．1 cDNA microarray could be used practically for monitoring the expression level of thousands of genes  53-54
  5．2 A repeated gene that could transcribed into RNA with polyA tail also existed in the structural gene of 23S rRNA in ricechloroplast genome  54-55
  5．3 Cytochrome C gene may play a role in stress response induced by ABA  55
Conclusion:  55-56
Reference  56-57

cDNA MICROARRAY MONITORING OF mRNA EXPRESSION PATTERN IN DIFFERENT RICE TISSUES AND STEM TISSUE INDUCED BY PHYTOHORMONE ABSCISIC ACID

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