黄瓜幼果cDNA文库构建与部分ESTs分析
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摘要
黄瓜(Cucumis sativus L．)原产喜马拉雅山南麓的印度北部地区，是一种世界性的重要蔬菜，为大众喜爱，有很高的营养价值。印度于3000年前开始栽培黄瓜。随着南亚民族的迁移和往来，黄瓜由原产地向东传播到中国南部，东南亚各国及日本等，在我国已有2000多年的栽培历史。黄瓜是喜温但不耐高温的一年生攀缘性草本植物，从播种出苗到采摘的时间较短，是最适合周年生产、均衡供应的一种瓜类。常用的植物遗传育种的原理与方法在提高黄瓜性状方面已失去了原有的优势，即长期通过表型值来估计育种值，使得性状的遗传进展越来越小。随着分子遗传学原理和生物技术方法的发展，从分子水平上对植物的生产性能进行改良有了可能，从而为植物分子育种打下基础。由于种内资源的有限，利用基因工程将是今后黄瓜品种改良中重组远缘有用基因的有效手段。对黄瓜基因进行研究，可为进一步的分子育种打下基础。黄瓜果实是其经济产品器官和食用部位，脆嫩、具有丰富的营养价值，它的生长发育过程、物质合成与积累、外部形态特征和内在品质性状等无不影响其产品器官的产量构成和经济价值。探讨黄瓜果实的生长发育特点，尤其是探讨黄瓜果实生长发育过程中的基因表达与调控方式，可以为今后改良黄瓜的结实习性、促进果实生长、提高经济产量和改善果实品质等方面奠定基础。本实验以黄瓜幼果为实验材料，以λTriplEx2为载体，构建了黄瓜cDNA文库，通过随机挑选部分克隆进行EST测序后，进行网上同源性比较和基因功能分析，得出以下结论：
一、黄瓜(Cucumis sativus L．)cDNA文库的构建
采用上海生工的Trizol试剂盒提取黄瓜的总RNA，1％琼脂糖凝胶电泳的结果显示：提取的黄瓜RNA的28s、18s、5s条带较为清晰，说明mRNA受分解的程度小。
西南农业大学硕士学位论文
采用Promega公司的PofyATtrac必mRNA Isolation systemm从RNA中分离提取
mRNA，经紫外分光光度计测定，mRNA纯度较高，能够满足实验的需要。
采用s以盯w 011即nucleotide和eosn仍’PeR Primer为引物，在助werscdPt侧
Reverse Tn江巧criPtase酶的作用下合成第一链cDNA并进一步合成第二链cDNA，经PCR产
物纯化和劝I酶切后用CHROMA SPIN礴00 colulnn柱进行分级分离以去除小于400bP的
cDNA片段和一些核糖体RNA，浓缩回收后按比例与载体人TriplExZ(带有劝酶切末端)连
接.采用stratagene公司的Gigapack⑧m Gofd一7 packaging extract试剂盒进行包装，获得
cDNA文库。
包装产物中加入终浓度为7%的DMSO，分装后置于一80℃保存。
二、文库质量的鉴定
将得到的cDNA原始文库按l:10的比例进行稀释，将文库的稀释液与XLI一Blue菌株
的感受态细胞混匀，加入0.器的顶层培养基以及IPTG和x~gal，倒平板并在37℃下过夜培养，
计算噬菌斑数目及蓝白噬菌斑数量。得出噬菌体cDNA文库的滴度为1.165xl咋五刀ml，重组
率94.42%。根据载体的多克隆位点，用反oRI和从”dlll进行双酶切，得到该文库的大多数
外源插入片段平均大小在50obP以上。采用相同方法测定cDNA扩增文库的滴度约为101。
Pfi材inl。
三、EST测序
从cDNA文库中随机挑选部分克隆，经及oRI和去打”dlll双酶切鉴定，进行1%琼脂糖
凝胶电泳，结果显示:大部分克隆都有外源片段的插入。本实验共测了141个阳性克隆，采
用人TriplExZ载体多克隆位点胡IA端的pTrips‘sequenei吧primer(CTcCGAGATcToGAc)
作为测序引物，即从。DNA的5‘端进行测序，去掉低质量EST序列后剩余139条，测序成
功率为98.58%，全长序列有36条，占25.53%。最长的EsT为706bP，平均长度562.74bp，
长度峰度在600bP左右，79.43%的EST长度集中在500~7oobPo
四、测序结果分析
从分析结果看，EST序列中有部分序列为同一基因。这些序列经clusta1W软件比较后拼
接形成一个新的序列。本实验共拼接出22个。139个EST经拼接共获得非重复序列116条，
占8145%。经BLASTx与美国国立卫生研究院国家生物信息学研究所(NCBI)的Ge血日出玄
摘要
公二二三二二留翻巴巴巴里曰吕曰皿
数据库(httP://认四切刃.ncbi.nlm.nih.gov)中现存的ESTs进行同源分析后表明，所得到的139
个ESTs序列中有133个在其中可以找到同源序列，其中97条(69.78%)与己知序列高度相
似，36条(25.90%)为低度相似序列，在GenBank中未找到匹配同源序列的ESTs为6个
(4.32%)。获得104个(74.82%)己知基因，6个(4.32%)新基因，29个(20.86%)未知
基因。在测定的134个ESTs中，核糖体蛋白基因ESTs最多为27个，占测定总数的19.42%。
Cucumber (Cucumis sativus L.) is a kind of important vegetable in the world. It is highly nutrient and popular with people. It originated from the northern part of India, south of the Hymalays. Cucumber had been cultivated in India 3000 years ago. With the migration of the south Asians, cucumber spreaded to the south of China, southeastern Asia and Japan etc., and has been cultivated in China for 2000 years. Cucumber is a kind of annual climber plant that likes to grow in warm condition. The interval between cucumber's seeding and harvest is so short that it is adaptable for all-year cultivation. The ovary is the edible part, crisp and alimental. The common theories and methods of plant genetic breeding had lost their advantage in improving physiological character. It means that the genetic improvement of character became smaller because of long-time evaluating the breeding value by phenotypic value. With the development of molecular genetics and biotechnology, there comes to possibility to improve
the plant physiological character on the molecule level. Owing to the limitation of inner-specific resource, gene project will be the effective way to recombine the useful genes in the improvement of cucumber breeding. The research of the cucumber genes can provide the foundation for further molecular breeding. It is easy to sample and analyse young fruit of cucumber that possess all genes in mature fruit. Using cucumber fruit as material and TriplEx2 as vector, Cucumber (Cucumis sativus L.) cDNA library has been constructed in our study. We selected and sequenced partial colones, compared the homology and analyzed the gene function by bioinformation method. 1. Construction of Cucumber (Cucumis sativus L.) cDNA library
The total RNA of Cucumber (Cucumis sativus L.) was extracted with Trizol kit, and the mRNA was extracted with PolyATtract (?)) mRNA Isolation SystemIII, the agar gel electrophoresis showed
the total RNA has three bands and the degree of purity could fit for the following experiments.
Using SMART IV Oligonucleotide and CDSHI/3' PCR Primer as primer, PowerScriptTM
Reverse Transcriptase as polymerase, first-strand cDNA has synthesized and following dscDNA
synthesized by primer LD-PCR. After PCR purification and sfi I digestion, small cDNA fragment
and some rRNA removed by CHROMA SPIN-400 column. Then cDNA fraction was ligated to
TriplEx2 vector and packaged , cDNA library was gotten.
Packaging product can be stored at -80℃ for at least one year in 7% DMSO.
2. Identifying the quality of cDNA library
1:10 dilution of the packaging extract was made and mixed with XL 1-Blue overnight culture, in which, 0.7% top agar, IPTG and x-gal were added. The mixture was poured onto 90mm LB/MgSO4 plates prewarmed to 37℃ and the plates were inverted and incubated at 37℃ overnight. By counting the plagues and calculating the ratio of white (recombinant) to blue (nonrecombinant) plaques, the titer of the phage was 1.165×106pfu/ml, and the recombinant percentage was 94.17%. Using EcoR I and HindIII digestion, the results show the length of inserting fragments were beyond 500bp. The titer of amplified library was about 1010 pfu/ml.
3. EST sequencing
Partial clones were selected from cDNA library, after EcoR I and HindIII. digestion and 1% agar gel electrophoresis, the result showed most of the clones had inserting fragment. In the experiment, 141 positive clones were sequenced with pTriP5 ' Sequencing Primer (CTCCGAGATCTGGAC) and the success ratio was 98.58%. 36 full-length sequences were obtained and the longest fragment was 706bp. 79.43% fragments gathered in the zone of 500~700bp.
4. Analysis of sequencing result
The analysis showed that some sequences encoded the same gene. After assembling by clustal W, we obtained 116 unigenes. Comparing the homology and analyzing the gene function by bioinformation method, we found 97 unigenes had significant similarities with the known EST, and the other 42 unigenes had low or no similarities with the known genes or EST sequences.
一、黄瓜(Cucumis sativus L．)cDNA文库的构建
采用上海生工的Trizol试剂盒提取黄瓜的总RNA，1％琼脂糖凝胶电泳的结果显示：提取的黄瓜RNA的28s、18s、5s条带较为清晰，说明mRNA受分解的程度小。
西南农业大学硕士学位论文
采用Promega公司的PofyATtrac必mRNA Isolation systemm从RNA中分离提取
mRNA，经紫外分光光度计测定，mRNA纯度较高，能够满足实验的需要。
采用s以盯w 011即nucleotide和eosn仍’PeR Primer为引物，在助werscdPt侧
Reverse Tn江巧criPtase酶的作用下合成第一链cDNA并进一步合成第二链cDNA，经PCR产
物纯化和劝I酶切后用CHROMA SPIN礴00 colulnn柱进行分级分离以去除小于400bP的
cDNA片段和一些核糖体RNA，浓缩回收后按比例与载体人TriplExZ(带有劝酶切末端)连
接.采用stratagene公司的Gigapack⑧m Gofd一7 packaging extract试剂盒进行包装，获得
cDNA文库。
包装产物中加入终浓度为7%的DMSO，分装后置于一80℃保存。
二、文库质量的鉴定
将得到的cDNA原始文库按l:10的比例进行稀释，将文库的稀释液与XLI一Blue菌株
的感受态细胞混匀，加入0.器的顶层培养基以及IPTG和x~gal，倒平板并在37℃下过夜培养，
计算噬菌斑数目及蓝白噬菌斑数量。得出噬菌体cDNA文库的滴度为1.165xl咋五刀ml，重组
率94.42%。根据载体的多克隆位点，用反oRI和从”dlll进行双酶切，得到该文库的大多数
外源插入片段平均大小在50obP以上。采用相同方法测定cDNA扩增文库的滴度约为101。
Pfi材inl。
三、EST测序
从cDNA文库中随机挑选部分克隆，经及oRI和去打”dlll双酶切鉴定，进行1%琼脂糖
凝胶电泳，结果显示:大部分克隆都有外源片段的插入。本实验共测了141个阳性克隆，采
用人TriplExZ载体多克隆位点胡IA端的pTrips‘sequenei吧primer(CTcCGAGATcToGAc)
作为测序引物，即从。DNA的5‘端进行测序，去掉低质量EST序列后剩余139条，测序成
功率为98.58%，全长序列有36条，占25.53%。最长的EsT为706bP，平均长度562.74bp，
长度峰度在600bP左右，79.43%的EST长度集中在500~7oobPo
四、测序结果分析
从分析结果看，EST序列中有部分序列为同一基因。这些序列经clusta1W软件比较后拼
接形成一个新的序列。本实验共拼接出22个。139个EST经拼接共获得非重复序列116条，
占8145%。经BLASTx与美国国立卫生研究院国家生物信息学研究所(NCBI)的Ge血日出玄
摘要
公二二三二二留翻巴巴巴里曰吕曰皿
数据库(httP://认四切刃.ncbi.nlm.nih.gov)中现存的ESTs进行同源分析后表明，所得到的139
个ESTs序列中有133个在其中可以找到同源序列，其中97条(69.78%)与己知序列高度相
似，36条(25.90%)为低度相似序列，在GenBank中未找到匹配同源序列的ESTs为6个
(4.32%)。获得104个(74.82%)己知基因，6个(4.32%)新基因，29个(20.86%)未知
基因。在测定的134个ESTs中，核糖体蛋白基因ESTs最多为27个，占测定总数的19.42%。
Cucumber (Cucumis sativus L.) is a kind of important vegetable in the world. It is highly nutrient and popular with people. It originated from the northern part of India, south of the Hymalays. Cucumber had been cultivated in India 3000 years ago. With the migration of the south Asians, cucumber spreaded to the south of China, southeastern Asia and Japan etc., and has been cultivated in China for 2000 years. Cucumber is a kind of annual climber plant that likes to grow in warm condition. The interval between cucumber's seeding and harvest is so short that it is adaptable for all-year cultivation. The ovary is the edible part, crisp and alimental. The common theories and methods of plant genetic breeding had lost their advantage in improving physiological character. It means that the genetic improvement of character became smaller because of long-time evaluating the breeding value by phenotypic value. With the development of molecular genetics and biotechnology, there comes to possibility to improve
the plant physiological character on the molecule level. Owing to the limitation of inner-specific resource, gene project will be the effective way to recombine the useful genes in the improvement of cucumber breeding. The research of the cucumber genes can provide the foundation for further molecular breeding. It is easy to sample and analyse young fruit of cucumber that possess all genes in mature fruit. Using cucumber fruit as material and TriplEx2 as vector, Cucumber (Cucumis sativus L.) cDNA library has been constructed in our study. We selected and sequenced partial colones, compared the homology and analyzed the gene function by bioinformation method. 1. Construction of Cucumber (Cucumis sativus L.) cDNA library
The total RNA of Cucumber (Cucumis sativus L.) was extracted with Trizol kit, and the mRNA was extracted with PolyATtract (?)) mRNA Isolation SystemIII, the agar gel electrophoresis showed
the total RNA has three bands and the degree of purity could fit for the following experiments.
Using SMART IV Oligonucleotide and CDSHI/3' PCR Primer as primer, PowerScriptTM
Reverse Transcriptase as polymerase, first-strand cDNA has synthesized and following dscDNA
synthesized by primer LD-PCR. After PCR purification and sfi I digestion, small cDNA fragment
and some rRNA removed by CHROMA SPIN-400 column. Then cDNA fraction was ligated to
TriplEx2 vector and packaged , cDNA library was gotten.
Packaging product can be stored at -80℃ for at least one year in 7% DMSO.
2. Identifying the quality of cDNA library
1:10 dilution of the packaging extract was made and mixed with XL 1-Blue overnight culture, in which, 0.7% top agar, IPTG and x-gal were added. The mixture was poured onto 90mm LB/MgSO4 plates prewarmed to 37℃ and the plates were inverted and incubated at 37℃ overnight. By counting the plagues and calculating the ratio of white (recombinant) to blue (nonrecombinant) plaques, the titer of the phage was 1.165×106pfu/ml, and the recombinant percentage was 94.17%. Using EcoR I and HindIII digestion, the results show the length of inserting fragments were beyond 500bp. The titer of amplified library was about 1010 pfu/ml.
3. EST sequencing
Partial clones were selected from cDNA library, after EcoR I and HindIII. digestion and 1% agar gel electrophoresis, the result showed most of the clones had inserting fragment. In the experiment, 141 positive clones were sequenced with pTriP5 ' Sequencing Primer (CTCCGAGATCTGGAC) and the success ratio was 98.58%. 36 full-length sequences were obtained and the longest fragment was 706bp. 79.43% fragments gathered in the zone of 500~700bp.
4. Analysis of sequencing result
The analysis showed that some sequences encoded the same gene. After assembling by clustal W, we obtained 116 unigenes. Comparing the homology and analyzing the gene function by bioinformation method, we found 97 unigenes had significant similarities with the known EST, and the other 42 unigenes had low or no similarities with the known genes or EST sequences.
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48．高冬玲，陈东．改良的一步热酚法及Trizol试剂盒提取组织中RNA的比较．开封医专学报．2000，19(4)：3-4．
49．王波，顾宏嫒．Wistar大鼠乳鼠晶体cDNA文库的构建．齐齐哈尔医学院学报．1996，17(3)：161-163．
50．姚知行．分子生物学技术讲座(三)——真核细胞mRNA的提取，纯化，分析及cDNA文库的构建．生物学通报．1997，32(8)：23-24．
51. Kato, S., Sekine, S., Oh, S.-W. et al. Construction of a human full-length cDNA bank. Gene. 1994,150:243-250.
52. D'Alession, J.M.&Gerard, G.E Second-strand cDNA synthesis with E.coli DNA polymerase Ⅰ and RNase H: the fate of information at the mRNA 5'terminus and the effect of E.coli DNA ligase. Nucleic Acid Res. 1988, 16:1999-2014.
53. Chenchik A, Zhu YY, Diatchenko L, et al. Generation and use of high-quality cDNA from small amounts of total RNA by SMART PCR [A]. Eds. Siebert, E D. Ln Gene Cloning and Analysis by RT-PCR[M]. USA: Biol Techniques Press, 1998,305-319.
54. Okayama, H.&Berg, P. High efficiency cloning of full-length cDNA. Mol.Cell.Bilo. 1982,2:161-170.
55．潘尚领，朱春江．3种PCR产物纯化方法的比较．广西医科大学学报．2001，18(5)：768-769．
56．赵微平．植物基因组：构建、表达和调控．首都师范大学出版社．1996．
56. Lorkovic ZJ, Schroder WP, Pakrasi HB et al. Molecular characterization of PsbW, a nuclear-encoded component of the photosystem Ⅱ reaction center complex in spinach. Proc Natl Acad Sci U S A. 1995 Sep 12, 92(19): 8930-4.
58．王忠，王三根．植物生理学．中国农业出版社．2000，27-29．
59. Lin CT, Lin MT, Chen YT et al. The gene structure of Cu/Zn-superoxide dismutase from sweet potato. Plant Physiol. 1995 Jun, 108(2):827-8.
60. Clark AM, Jacobsen KR, Bostwick DE et al. Molecular characterization of a phloem-specific
gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima. Plant J. 1997 Jul, 12(1):49-61.
61. Goldmark P, Curry L A, Morris C et al. Cloning and expression of an embryo specific mRNA up-regulated in hydrated dormant seeds. Plant Mol. Bilo. 1992,19:257-263.
62. Johnson R R, Cranston H J, Chaverra M E et al. Characterization of cDNA clones for differentially expressed genes in embryos of dormant and nondormant Arena fatua L. earyopses, Plant Mol. Biol.1995, 28:113-122.
63．崔涛，肖杰．DNA结合蛋白质的结构与功能．生命的化学．1990，10(2)：1-4．
64. Ohta O, Sugita M and Sugiura M. Three types of nuclear genes encoding chloroplast RNA-binding proteins (cp29, cp31, and cp33) are present in Arabidopsis thaliana: presence of cp31 in chloroplasts and its homologue in nuclei/cytoplasms. Plant Mol.Biol. 1995,27:529-539.
65. Salts Y, Wachs R, Gruissem W et al. Sequence coding for a novel proline-rich protein preferentially expressed in young tomato fruit. Plant Mol.Biol. 1991,17:149-150
66. Salts Y, Keingsbuch D, Wachs R et al. DNA sequence of the tomato fruit expressed proline-rich protein gene TPRP-F1 reveals an intron within the 3 untranslatedtranscript. Plant Mol.Biol.1992, 18:407-409.
67. Ueoka-Nakanishi H, Nakanishi Y, Tanaka Y. Properties and molecular cloning of Ca2+/H+ antiporter in the vacuolar membrane ofmung bean. Eur J Biochem. 1999 Jun, 262(2):417-25.
68. O'Neill SD, Zheng CC. Abundance of mRNAs encoding HMG1/HMG2 class high-mobility-group DNA-binding proteins are differentially regulated in cotyledons of Pharbitis nil. Plant Mol Biol. 1998 May, 37(2):235-41.
69. Gensehik P, Parmentier Y, Durr A et al. U-biquitin genes are differentially regulated in protoplast-dedved cultures of Nicotiana sylvestds and in response to various stresses. Plant Mol Biol. 1992 Dec, 20(5):897-910.
70. Bahrami AR, Gray JE. Expression of a proteasome alpha-type subunit gene during tobacco development and senescence. Plant Mol Biol. 1999 Jan, 39(2):325-33.
71. Baiges I, Schaffner AR, Mas A. Eight cDNA encoding putative aquaporins in Vitis hybrid Riehter-110 and their differential expression. J Exp Bot. 2001 Sep, 52(362): 1949-51.
72. Bouvier-Nave P, Husselstein T, Benveniste P. Two families of sterol methyltransferases are involved in the first and the second methylation steps of plant sterol biosynthesis. Eur J Biochem. 1998 Aug 15, 256(1):88-96.
73. Huang J, Struck F, Matzinger DF et al. Flower-enhanced expression of a nuclear-encoded mitochonddal respiratory protein is associated with changes in mitochondrion number. Plant Cell. 1994 Mar, 6(3):439-48.