水稻窄卷叶突变体nrl4的表型分析与基因定位

Phenotype Analysis and Gene Mapping of Narrow and Rolling Leaf Mutant nrl4 in Rice(Oryza sativa L.)

【目的】研究水稻卷叶突变体叶形变化的分子机理,鉴定出新的水稻卷叶基因。【方法】利用EMS诱变籼稻品种浙农34,获得一个窄卷叶突变体,命名为nrl4(narrow and rolling leaf 4)。在抽穗期随机选取野生型浙农34和nrl4各10株,对其进行表型观察和主要农艺性状调查等,并测定叶绿素含量;同时用Zeiss荧光显微镜观察剑叶中部叶片横切面维管束的数目并统计泡状细胞数量。以多代自交稳定的突变体nrl4为母本与野生型浙农34杂交,观察植物F_1和F_2叶片表型,统计F_2中性状分离比并作卡方测验,分析突变表型的遗传行为。利用nrl4与粳稻品种浙农大104杂交,采用F_2分离群体进行基因精细定位。利用定量表达对定位区间内5个预测的基因进行相对表达量的分析。【结果】与野生型相比,窄卷叶突变体nrl4抽穗期时全部叶片卷曲且变窄、叶绿素含量升高;株高稍有增加,结实率增大,籽粒明显变长、变窄。窄卷叶突变体nrl4功能叶夹角不同程度减小,叶形更为直立。突变体叶近轴表面特有的泡状细胞数目降低、体积变小,导致叶片向内卷曲。突变体nrl4的叶脉数减少,叶片变窄,中脉一侧2个大维管束之间的小维管束平均为4.5个,而野生型为6.0个。遗传分析表明,突变体nrl4和浙农34杂交的F_1表型正常,F_2群体中正常植株与窄卷叶突变植株的分离比符合3﹕1,表明突变体nrl4的突变性状受1对隐性核基因控制;利用SSR和In Del分子标记将nrl4定位在水稻第3染色体长臂3M11103和3M1115之间、物理距离约为53 kb的区间。在这一区间内,有5个预测注释基因,序列比对和表达分析表明在野生型浙农34和突变体nrl4之间,这些基因序列及启动子序列均未发生变化,但是LOC_Os03g19770在突变体叶片中的表达量显著增加。【结论】nrl4叶片变窄与维管束数目减少有关,叶片发生内卷与泡状细胞数目减少、体积变小有关。水稻窄卷叶突变体nrl4的性状由1对隐性核基因控制,该基因位于第3染色体In Del标记3M11103和3M1115物理距离约为53 kb的区间内,预测区间内基因序列及5′UTR区未发现碱基变异,但LOC_Os03g19770在突变体叶片中的表达量达到了野生型植株叶片的17.5倍,推测LOC_Os03g19770为候选基因。

[Objective] Rice leaf mutant was used to study molecular mechanisms of leaf traits, and to identify the related novel rolling genes in rice. [Method] The mutant with narrow and rolling leaves was derived from the indica cultivar Zhenong34 induced by ethyl methylsulphonate （EMS）, named nrl4. At heading stage, nrl4 and WT Zhenong 34 were randomly selected 10 strains to measure the main agronomic traits and chlorophyll content of nrl4 and WT were tested at the same time. The bulliform cells were observed and counted as well as the number of large and small veins in transverse section of blade under the Zeiss microscope. The leaf phenotype of the F1 plants and F2 population which derived from the crossing of nrl4 with Zhenong 34 were investigated and the segregation ratio of normal and rolling leaves were analyzed by chi-square test in the F2 population. The F2 population from crossing of nrl4 with Zhenong 104 was used for genetic analysis and gene fine mapping. Five genes in the located region were analyzed by gene quantitative expression. [Result] Morphological analysis showed that all leaves of mutant nrl4 were narrow and rolling. In addition, compared with wild type Zhenong 34, plant height, seed setting rate in main panicle and pigment content of mutant nrl4 were increased, as well as grain length of nrl4, but the width of grain was decreased. Leaf angles of fianctional leaves were all decreased leading to more erecting plant type. Statistical analysis suggested that the rolling leaf phenotype might be caused by the change of number and size of bulliform cells which especially existed at the adaxial side of blade; moreover, in accordance with reduced leaf blade width, leaves of nrl4 contain a decreased number of large veins and small veins. There were 6.0 small veins between two large veins on one side of main vein averagely in mutant nrl4 leaf while there were 4.5 in wild type Zhenong34. Genetic analysis indicated that the mutant trait was controlled by a single recessive gene, the gene nrl4 was located in a confined region of 53 kb flanked by two InDel makers 3M11103 and 3M1115 on the long arm of chromosome 3, where five annotated genes were predicted. Based on the result of sequencing, there was no mutation occurred in the gene sequence and promoter sequence of these predicted genes, but strong changes in gene expression pattern of LOC_OsO3gl9770 according to the real time quantitative PCR. These results are very valuable for further study on this gene. [ Conclusion] The narrow leaves are related to reduced number of vasculars, moreover the rolling blade of mutant nrl4 might resulted from the decreased area and number of bulliform cells. The mutant nrl4 is controlled by a single recessive nuclear gene, which is located on chromosome 3, between 3M 11103 and 3M1115 with a physical distance of 53 kb. No nucleotide sequence mutation was found to occur in the gene sequence or the 5＇UTR of all annotated genes, but the expression of LOC_OsO3gl9770 is strongly promoted in mutant nrl4, which is 17.5 times of wild type and it may be the candidate gene.