论文标题:玉米和黑麦草漆酶基因的克隆和系统发育分析及玉米水分胁迫下基因表达研究
Cloning and Phylogenetic Analysis of Laccases in Maize and Ryegrass and Gene Expression of Water Stress in Maize
论文作者 路运才
论文导师 王天宇；黎裕,论文学位 博士,论文专业 作物遗传育种
论文单位 中国农业科学院,点击次数 201,论文页数 139页File Size18939k
2004-09-01论文网 http://www.lw23.com/lunwen_3399697/ 玉米;漆酶;黑麦草;分子进化;基因克隆;cDNA-AFLP;水分胁迫;基因表达
Maize; Laccase; Ryegrass; Molecular evolution; Gene cloning; cDNA-AFLP;Water stress; Gene expression
目前,玉米已经成为超过小麦的第二大粮饲兼用作物。消化率和干旱是影响玉米饲用品质和产量的两个重要因素。常规育种在饲用品质改良和抗旱育种中发挥了重要作用,但该途径费时费力,效率不高。生物技术的迅猛发展,为在分子水平上开展玉米饲用品质改良和抗旱性分子机制研究提供了有利契机。 本研究利用基因同源序列克隆技术、cDNA-AFLP技术和生物信息学技术,一方面,进行黑麦草和玉米漆酶基因的克隆与分析;另一方面,对水分胁迫条件下4个玉米自交系材料的基因表达情况进行深入研究,确定与玉米抗旱相关的功能基因,从而为遗传工程改良饲料作物的消化率和增强玉米种质的抗旱性提供重要的物质基础和理论依据。这对于我国农业种植结构调整和畜牧业的发展以及实施高效节水农业均具有一定的理论和实践意义。 本研究通过对参与木质素合成途径中一个重要酶-漆酶的系统分析与克隆以及利用cDNA-AFLP技术对不同抗旱性的玉米种质在水分胁迫条件下的基因表达规律进行了研究,获得了以下主要结果: 1.以64个不同来源的植物漆酶为材料,对其氨基酸序列进行了比较分析,并进行了分子进化机制的探讨。发现漆酶作为蓝铜氧化酶蛋白家族的成员,具有一个或数个不等的保守区域(HWHG和HLHG)及N糖基化结合位点。系统发生关系表明,植物漆酶是一个高度趋异的多基因家族,在种子植物分化为被子植物和裸子植物的前后,均可能发生了基因复制; 2.进一步通过对64个漆酶的一系列特征进行分析发现:同一物种的不同漆酶成员在序列上差异很大;在单子叶和双子叶植物中均有细胞外和细胞内漆酶蛋白;糖基化位点数目(8-23个)和等电点值的变化范围都很大(5-10);漆酶的催化底物广泛;在不同组织中差异表达。因此,植物漆酶除了参与木质素的生物合成外,还可能具有其它功能; 3.在27个水稻漆酶基因中,有9个位于第9号染色体上,6个位于第11号染色体上。这就意味着超过一半的漆酶成员集中在2条染色体上。水稻第1号染色体上的漆酶基因分散在3个不同的区段。从GRLC26到GRLC1跨越了21Mb。GRLC26位于区段1(15371897-15374072 bp);GRLC6位于区段2(25214631-25216628 bp)。区段3为GRLC5和GRLC1之间(1.3Mb),该区域位于C126435和C15960标记之间。位于第11号染色体上的漆酶基因。聚集于3个区段。其中,GRLC8、GRLC11、GRLC12、GRLC15位于短臂末端。我们发现GRLC11、GRLC12和GRLC15与水稻的一个MYB转录因子的基因克隆在染色体上的位置相近。17个拟南芥漆酶基因只有AtLC1和AtLC7分布于第1、4号染色体,其余则密集分布于第2、5号染色体。依据已经发表的拟南芥基因组部分复制区域,我们发现在第3号染色体的AtLC7和第5号染色体的AtLC8、AtLC9、AtLC10、AtLC11和AtLC12之间发生了基因复制; 4.拟南芥16个漆酶基因的数字化Northern分析结果显示,多数基因在根和茎组织强烈表
Maize is the second largest crop for food and feed in China. Forage quality and yield of maize are greatly affected by digestibility and drought stress. Conventional maize breeding has played an important role in improving forage quality and drought tolerance, but it is time-consuming and labor intensive. The rapid progress in molecular biology and genomics has provided an opportunity in accelerating forage improvement and dissecting mechanism of drought tolerance.In the present study, the laccase genes in ryegrass and maize were cloned and compared, while the gene expression of four maize inbred lines under water stress were analyzed, based on gene homology sequence cloning, cDNA-AFLP and bioinformation techniques. This work may provide theoretical and practical guides for accelerating and facilitating breeding for good forage quality and drought tolerance in maize and thus contribute to the structural adjustment of agricultural production and sustainable agriculture in China. The main results are as follows:1. A comprehensive phylogenetic analysis of 64 laccase genes from monocots, dicots and gymnosperm was conducted to identify putative orthologues and paralogues within the multiple-gene family. By multisequence alignments, a wide range of conserved domains and motifs were found in most members. Phylogenetic analysis indicated that plant laccase genes are a highly divergent multigene family. Duplication of laccase genes has been a continuous process occurring before and after the seed plants diverged into gymnosperms, monocots, and dicots.2. Plant laccases from different plant species, and in some cases, even within the same species, had a relatively low level of sequence homology. Both extracelluar and intracellular plant laccases were found.The range of pI values of plant laccase, substrate specificity, and numbers of N-glycosylation site varied greatly. Furthermore, plant laccase genes were confirmed to be of tissue-specific expression, inferring that this enzyme group is likely to carry out diverse functions in the plant.3. A total of 27 and 17 laccase genes from two model plants, rice and Arabidopsis. respectively, were used to construct frame maps. Of the 27 laccase genes from rice, nine were located on chromosome 1 and 6 on chromosome 11, indicatingthat more than half of the members concentrated on two chromosomes. Laccase genes mapped on rice chromosome 1 were dispersed into three regions that spanned 21Mb estimated from GRLC26 to GRLC1. GRLC1 was located around region 1 (15371897-15374072 bp) whereas GRLC6 was located around region 2 (25214631- 25216628 bp). Region 3 ranged from GRLC5 to GRLC1 (1.3 Mb), which spanned between C126435 and C15960 according to the published data on chromosome 1. On chromosome 11, the laccase genes also were divided into three regions. Four of them were located at the distal region of short arm. Comparing with the published map of chromosome 11 based on a doubled haploid population, it was found that GRLC11,12 and 15 were close to a gene cloned from rice, designated as a MYB transcription factor. Of the 17 Arabidopsis laccase genes, only AtLCl and AtLC7 were dispersed on chromosomes 1 and 4,
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