欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2009, Vol. 35 ›› Issue (9): 1576-1583.doi: 10.3724/SP.J.1006.2009.01576

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜种子不同发育时期SSH文库的构建

彭琦1,胡燕1,杜培粉1,2,谢青轩1,2,阮颖1,2,*,刘春林1,*   

  1. 1作物种质创新和资源利用国家重点实验室培育基地；2湖南农业大学生命科学技术学院，湖南长沙410128
  • 收稿日期:2009-01-02 修回日期:2009-04-26 出版日期:2009-09-12 网络出版日期:2009-07-03
  • 通讯作者: 刘春林，E-mail: liucl100@126.com; 阮颖，E-mail: yingruan@hotmail.com; Tel: 0731-4635294
  • 基金资助:

    本研究由国家重点基础研究发展计划（973计划）项目（206CB101603）资助。

Construction of SSH Library with Different Stages of Seeds Development in Brassica napus L.

PENG Qi1,HU Yan1,DU Pei-Fen1,2,XIE Qing-Xuan1,2,RUAN Ying1,2,*,LIU Chun-Lin1*   

  1. 1Pre-State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops;2College of Bio-Science and Technology,Hunan Agricultural University,Changsha,410128 China
  • Received:2009-01-02 Revised:2009-04-26 Published:2009-09-12 Published online:2009-07-03
  • Contact: LIU Chun-Lin，E-mail: liucl100@126.com;RUAN Ying，E-mail: yingruan@hotmail.com; Tel: 0731-4635294

PDF

4648

被引次数

46

摘要：

利用抑制差减杂交技术构建了20 d和35 d甘蓝型油菜湘油15发育种子特异表达基因的SSH文库。随机挑取单菌落进行PCR表明，文库质量较好。在20 d和35 d SSH文库中随机挑选489个阳性克隆进行测序，获得452条高质量的表达序列标签(EST)。对序列进行Blast比对及功能注释，比较20 d和35 d SSH文库的基因表达谱，发现在20 d SSH库中参与糖代谢的基因出现频率较高，而在35 d SSH库中与脂肪酸储存有关的油体蛋白家族、与脂肪酸转运有关的柠檬酸合酶、与脂肪酸合成有关的酰基载体蛋白去饱和酶等，参与油脂代谢相关基因出现频率较高。该结果对进一步研究油菜脂肪酸代谢调控的分子机制打下了基础。

关键词: 甘蓝型油菜, 种子发育, 脂肪酸, 抑制差减杂交, cDNA文库

Abstract:

Mechanism of fatty acid metabolic is a significant research topic in rapeseed molecular breeding. There are six hundreds genes and ESTs associated with fatty acid metabolism, 14% of which are conformed to participate in acrylic-fatty acid metabolism, 86% of which are speculated on sequences similarity and conservative domain with other species. But compared to the situation in Arabidopsis thaliana, molecular regulation mechanism of fatty acid metabolism in rapeseed has been less reported. In harvested rapeseed seeds, there is difference in seed fatty acid components among different varieties or the same variety grown under different ecological conditions. To further explore the molecular mechanism of fatty acid metabolic regulation of Brassica napus L., we investigated the assimilation product transition during the seed development. The starch reached a peak content at 20 days after pollination (20DAP) and was used up quickly after 20DAP, immediately the fatty acids content rapidly increased from 30DAP to 35DAP. According to the results, 20DAP developing seeds and 35DAP developing seeds were chosen for suppression subtractive hybridization (SSH), which is an effective tool for picking out specific expression genes among different samples. Two libraries, 20DAP SSH library derived from 20DAP seed cDNA as tester and 35DAP seed cDNA as driver and 35DAP library from 20DAP seed cDNA as driver and 35DAP seed cDNA as tester were constructed. The two SSH libraries had a high quality with high suppression subtractive efficiency after tested by PCR and RT-PCR. A total of 489 clones were randomly selected from the two libraries for sequencing and 452 high quality sequences tags were obtained. Blast analysis and functional annotation showed that most of the genes in 20DAP SSH library were relative to carbohydrate metabolism, while those in 35DAP library relative to fatty acid metabolic regulation. Significantly, 5 function-unknown genes in 20DAP library and 7 in 35DAP library were found out. In summary, this work adds an extra layer of complexity to the regulation of starch-to-oil transition and at the same time the different genes, especially the function-unknown genes shed light on studies of molecular mechanism of fatty acid metabolic regulation in seeds of Brassica napus L.

Key words: Brassica napus L., Seeds development, Fatty acid, Suppression subtractive hybridization(SSH), cDNA library

[1] DiatchenkoL, LauY F, CampbellA P, ChenchikA, MoqadamF, HuangB, LukyanovS, LukyanovK, GurskayaN, SverdlovE D, SiebertP D. Suppression subtractive hybridization: A method for generating differentially regulated or tissue specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93: 6025-6030

[2] Diatchenko L, Lukyanov S, Lau Y F, Siebert P D. Suppression subtractive hybridization: A versatile method for identifying differentally expressed genes. Methods Enzymol, 1999, 303: 349-380

[3] Siebert P D, Chenchik A, Kellogg D E, Lukyanov K A, Lukyanov S A. An improved PCR method for walking in uncloned genomic DNA. Nucl Acids Res, 1995, 23: 1087-1088

[4] Lukyanov K A, Launer G A, Tarabykin V S, Zaraisky A G, Lukyanov S A. Inverted terminal repeats permit the average length of amplified DNA fragments to be regulated during preparation of cDNA libraries by polymerase chain reaction. Anal Biochem, 1995, 229: 198-202

[5] Lambe T, Finlay D, Murphyand M,Martin F. Differential expression of connexin 43 in mouse mammary cells. Cell Biol Intl, 2006, 30: 472-479

[6] Sangrado-Vegas A, Lennington J B, Smith T J. Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridization. Cytokine, 2002, 17: 66-70

[7] Li R J, Wang H Z, Mao H, Lu Y T, Hua W. Identification of differentially expressed genes in seeds of two near-isogenic Brassica napus lines with different oil content. Planta, 2006, 224: 952-962

[8] Li X-Y(李小艳), Zhao Y(赵云), Zhou Y-T(周云涛), Li Y-Y(李熠毅), Wang M-L(王茂林). Construction and primary analysis of suppression subtractive library of dwarf mutant ‘NDF-1’in Brassica napus. Chin J Oil Crop Sci (中国油料作物学报), 2006, 28(4): 396-402(in Chinese with English abstract)

[9] Liu Y(刘阳), Wang M-L(王茂林), Qiu F(邱峰), Zhou Y-T(周云涛), Zhao Y(赵云), Zhang F(张帆). The construction of the suppression subtractive library of the Brassica napus mutant Cr3529. J Sichuan Univ (四川大学学报), 2005, 42(5): 1029-1032(in Chinese with English abstract)

[10] Ohlrogge J B, Kuhn D N, Stumpf P K. Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad Sci USA, 1979, 76: 1194-1198

[11] Ohlrogge J, Browse J. Lipid biosynthesis. Plant Cell, 1995, 7: 957-970

[12] Thelen J J, Ohlrogge J B. Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng, 2002, 4: 12-21

[13] Beisson F, Koo A J, Ruuska S, Schwender J, Pollard M, Thelen J J, Paddock T, Salas J J, Savage L, Milcamps A, Mhaske V B, Cho Y, Ohlrogge J B. Arabidopsis genes involved in acyl lipid metabolism: A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database. Plant Physiol, 2003, 132: 681-697

[14] Huang A H C. Oleosin and oil bodies in seeds and other organs. Plant Physiol, 1996, 110: 1055-1061

[15] Murphy D J, Vance J. Mechanisms of lipid-body formation. Trends Biochem Sci, 1999, 24: 109-115

[16] O'Hara P, Slabas A R, Fawcett T. Fatty acid and lipid biosynthetic genes are expressed at constant molar ratios but different absolute levels during embryogenesis. Plant Physiol, 2002, 129: 310-320

[17] Ohlrogge J, Pollard M, Bao X, Focke M, Girke T, Ruuska S, Mekhedov S, Benning C. Fatty acid synthesis: From CO2 to functional genomics. Biochem Soc Trans, 2005, 28: 567-573

[18] Morgunov I, Srere P A. Interaction between citrate synthase and malate dehydrogenase: Substrate channeling of oxaloacetate. J Biol Chem, 1998, 273: 29540-29544

[19] Pracharoenwattana I, Cornah J E, Smith S M. Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination. Plant Cell, 2005, 17: 2037-2048 Koyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata D. Over expression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil. Plant Cell Physiol, 2000, 41: 1030-1037
[1] 陈松余, 丁一娟, 孙峻溟, 黄登文, 杨楠, 代雨涵, 万华方, 钱伟. 甘蓝型油菜BnCNGC基因家族鉴定及其在核盘菌侵染和PEG处理下的表达特性分析[J]. 作物学报, 2022, 48(6): 1357-1371.
[2] 秦璐, 韩配配, 常海滨, 顾炽明, 黄威, 李银水, 廖祥生, 谢立华, 廖星. 甘蓝型油菜耐低氮种质筛选及绿肥应用潜力评价[J]. 作物学报, 2022, 48(6): 1488-1501.
[3] 袁大双, 邓琬玉, 王珍, 彭茜, 张晓莉, 姚梦楠, 缪文杰, 朱冬鸣, 李加纳, 梁颖. 甘蓝型油菜BnMAPK2基因的克隆及功能分析[J]. 作物学报, 2022, 48(4): 840-850.
[4] 黄成, 梁晓梅, 戴成, 文静, 易斌, 涂金星, 沈金雄, 傅廷栋, 马朝芝. 甘蓝型油菜BnAPs基因家族成员全基因组鉴定及分析[J]. 作物学报, 2022, 48(3): 597-607.
[5] 王瑞, 陈雪, 郭青青, 周蓉, 陈蕾, 李加纳. 甘蓝型油菜白花基因InDel连锁标记开发[J]. 作物学报, 2022, 48(3): 759-769.
[6] 张艳波, 王袁, 冯甘雨, 段慧蓉, 刘海英. 棉籽油分和3种主要脂肪酸含量QTL分析[J]. 作物学报, 2022, 48(2): 380-395.
[7] 石磊, 苗利娟, 黄冰艳, 高伟, 张忠信, 齐飞艳, 刘娟, 董文召, 张新友. 花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答[J]. 作物学报, 2021, 47(9): 1703-1711.
[8] 薛晓梦, 吴洁, 王欣, 白冬梅, 胡美玲, 晏立英, 陈玉宁, 康彦平, 王志慧, 淮东欣, 雷永, 廖伯寿. 低温胁迫对普通和高油酸花生种子萌发的影响[J]. 作物学报, 2021, 47(9): 1768-1778.
[9] 王艳花, 刘景森, 李加纳. 整合GWAS和WGCNA筛选鉴定甘蓝型油菜生物产量候选基因[J]. 作物学报, 2021, 47(8): 1491-1510.
[10] 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798.
[11] 唐鑫, 李圆圆, 陆俊杏, 张涛. 甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究[J]. 作物学报, 2021, 47(5): 983-990.
[12] 周新桐, 郭青青, 陈雪, 李加纳, 王瑞. GBS高密度遗传连锁图谱定位甘蓝型油菜粉色花性状[J]. 作物学报, 2021, 47(4): 587-598.
[13] 李书宇, 黄杨, 熊洁, 丁戈, 陈伦林, 宋来强. 甘蓝型油菜早熟性状QTL定位及候选基因筛选[J]. 作物学报, 2021, 47(4): 626-637.
[14] 张春, 赵小珍, 庞承珂, 彭门路, 王晓东, 陈锋, 张维, 陈松, 彭琦, 易斌, 孙程明, 张洁夫, 傅廷栋. 甘蓝型油菜千粒重全基因组关联分析[J]. 作物学报, 2021, 47(4): 650-659.
[15] 唐婧泉, 王南, 高界, 刘婷婷, 文静, 易斌, 涂金星, 傅廷栋, 沈金雄. 甘蓝型油菜SnRK基因家族生物信息学分析及其与种子含油量的关系[J]. 作物学报, 2021, 47(3): 416-426.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2