【作者】 于凤池；

【导师】 汪俏梅；

【作者基本信息】 浙江大学 ， 蔬菜学， 2003， 硕士

【摘要】 茎在植物体内水分、无机盐和有机物质的运输中发挥功能，并对水稻的产量和品质具有重大的决定作用。为了对水稻茎中基因表达谱和茎特异基因有一更好地了解，我们以水稻4—5叶期茎为实验材料构建cDNA文库，并对其进行EST测序和生物信息学分析，发现一些高丰度表达基因，为验证这些基因是否为茎特异性基因，选取9个EST克隆为探针，与容量为755个不同水稻表型的RNA微阵列杂交。本研究主要结果如下： 1．从水稻茎cDNA文库中随机挑取4704个克隆进行3’端测序，共获得3652条可读序列，经生物信息学分析，共获得2683条有效ESTs，并对其中2485条3’端具Poly(A)尾的ESTs作进一步分析；另外2630条序列已被GenBank收录。 2．本研究利用自主开发的分析软件DNAuser，结合人工精确判断，对2485条具有Poly(A)尾的序列进行了冗余度分析，共获得1903条独立基因，占全部序列的76.6％。其中低丰度表达基因1591条，占独立基因总数的83.6％；中丰度表达基因282条，占独立基因总数的14.8％；高丰度表达基因30条，占独立基因总数1.6％，其中最高的包含21个ESTs。说明在水稻茎组织中表达的基因绝大部分呈中、低丰度表达。 3．1903条独立基因与GenBank水稻数据库进行BLASTn比对，首次建立了水稻茎组织特异表达基因数据库。通过对数据的分析发现，新基因1066条，占56.0％；功能已知基因353条，占18.5％；有染色体定位信息的基因593条，占31.2％；既有功能注释又有染色体定位的基因109条，占5.7％。说明水稻茎组织中大部分基因为新基因，意味着大量基因可能在水稻茎组织中特异表达。 4．结合拟南芥基因功能分类方法，将353条有功能注释基因划分为11大类：蛋白合成和降解基因(PRO,21.6％)、代谢基因(MET,17.3％)、能量基因(ENG，13.5％)、逆境反应基因(RES、12.9％)、信号转导基因(COM,8.3％)、功能分类不明确基因(MIS,7.6％)、转录基因(SCR,7.6％)、细胞结构相关基因(BIO、5.5％)、细胞生长、分裂和DNA合成基因(GRO,1.3％)、细胞内运输基因(TRA、4.3％)和发育基因(DEV、0.1％)。 5．在高丰度表达且有功能描述的基因中，发现叶绿体a/b结合蛋白(light harvesting chlorophyll a/b-binding protein)基因表达丰度最高，表达频率为21，其次为伸长因子(EF-lalpha)和脱落酸和逆境诱导蛋白(abscisic acid and stress 。inducible protein人表达频率均为 14。另外有富脯氨酸蛋白中roline ich protein，PRPRP）、微管蛋白（alpha－帅＂lin）、月类转t蛋白（hpid transfer protein， LTPL高度移动性群体蛋白伪igh mobility gTOUp protein，HMG）和苯丙氨酸 解氨酶（phenylalanine ammonia－lyase，PAL）等 20个基因。6．RNA微阵列杂交结果表明，高度移动性群体蛋白、伸长因子和过氧化氢酶存 在共调控表达；高度移动性群体蛋白在各表型中表达变化波动不大，是看家 基因；脂类转运蛋白和富脯氦酸蛋白的基因表达谱具很大的相似性；叶绿素 a／b结合蛋白在叶中高丰度表达且受光诱导，是与光合作用相关基因。各基因 在水稻不同部位（根、茎、叶）基因表达情况分析表明，微管蛋白在茎和根 中都具有较高的表达丰度，说明根的纤维化和茎的木质化是同一生化过程； 脱落酸和逆境诱导蛋白在茎中高丰度表达，易于为环境因子所诱导，在茎中 不能组成型稳定表达，不能认为是茎中特异性基因；富脯氨酸蛋白在茎中具 有较高的表达丰度；叶绿素ajb结合蛋白在茎中高丰度表达，且具有一定的 光节律现象，说明茎也是光合作用的主要器官，且茎中可能有其特异的叶绿 素ajb结合蛋白。更多还原

【Abstract】 Rice stem functions in the transportation of water, minerals and organic compounds, and is essential for the yield and quality of rice. We constructed cDNA library for rice stems at the stage of 4-5 leaves, and conducted EST sequencing and bioinformatical analysis for better understanding of gene expression pattern and stem-specific genes. Some highly-expressed genes were isolated in rice stem cDNA library, and nine cDNA probes were used for hybridization with RNA array containing 755 rice bio-phenotypes to test whether these genes were stem-specific or not. The main results were as follow:1. 4704 clones were randomly selected from rice stem cDNA library and sequenced from 3’-end by MegaBace 1000, 3652 of which produced readable sequences, and 2683 effective ESTs were isolated by bioinformatical analysis. Among them, 2485 high quality ESTs with poly (A)+ at 3 end were further analyzed, while additional 2630 ESTs have been registered in dbEST (http://www.ncbi.nlm.nih.gov/dbEST).2. 1903 unique genes were identified by redundancy analysis with DNA user software from 2485 3’-ESTs. The expression abundance of the 1903 unique genes was different in rice stem. 1591(83.6%) of them were low expression abundance genes, 282 (14.8%) of them were moderate expression abundance genes, and only 30 (1.6%) of them were high expression abundance genes in which the highest one consisting of 21 ESTs. The results indicated that most genes in rice stem were low or moderate expression abundance genes.3. 1903 ESTs were blasted with rice database of NCBI and the uni-gene database of rice stem were constructed. On the basis of database searches, 1066 (56.0%) were novel genes that had no homology in public database, 353 (18.5%) were known genes with function annotation or putative function annotation, 593 (31.2%) were genes with the information of location, and another 109 (5.7%) were genes with information of function and location. The results indicated that most genes in rice stem were unknown genes which express specifically in stem.4. Genes with function annotation or putative function annotation were assigned to 11 functional gene classes by the means of function classification in Arabidopsis. They are genes related to protein synthesis and destination (PRO, 21.6%), metabolism (MET, 17.3%), energy (ENG, 13.5%), cell rescue, defense, cell death and ageing (RES, 12.9%), cellular communication/signal transduction (COM,8.3%), transcription (SCR, 7.6% ), cellular biogenesis and organization (BIO, 5.5%), cell growth, cell division and DNA synthesis (GRO, 1.3%), transport facilitation and mechanism (TRA, 4.3%) and development (DEV, 0.1%) and miscellaneous: unclassified or unknown gene (MIS, 7.6%);5. 20 genes with high expression abundance and function annotation were identified in our study. Among them, light harvesting chlorophyll a/b-binding protein had the highest expression abundance, with the expression frequency of 21, while EF-1 alpha and abscisic acid and stress -inducible protein was the second, with the expression frequency of 14. Some other genes, including proline rich protein (PRP), alpha-tubulin, lipid transfer protein (LTP), high mobility group protein (HMG) and phenylalanine ammonia-lyase (PAL) were also identified.6. RNA array analysis showed that co-regulation existed in the expression among high mobility group protein, elongation factor-1 alpha and catalase. The expression of high mobility group protein changed little in all the bio-phenotypes and it seemed to be house-keeping gene. The expression profiles of lipid transfer protein and proline-rich protein were similar. Chlorophyll a/b-binding protein was highly expressed in leaves and induced by light, and it seemed to be a gene related to photosynthesis. Studies of gene expression in rice leaf, stem and root showed that tubulin was highly express both in stem and root, indicating that root fibrosis and stem lignification share the same chemical process. Abscisic acid-an更多还原