水稻黄叶突变体yl(t)的鉴定与遗传分析  

Identification and Genetic Analysis of the Yellow Leaf Mutant yl(t)in Rice(Oryza sativa)

作　　者：康伟伟 李哲理 易自力[2] 孙志忠[1] 盛夏冰[1,2] 黄安平 段美娟[3] 谭炎宁[1] KANG Wei-Wei;LI Zhe-Li;YI Zi-Li;SUN Zhi-Zhong;SHENG Xia-Bing;HUANG An-Ping;DUAN Mei-Juan;TAN Yan-Ning(State Key Laboratory of Hybrid Rice,Hunan Hybrid Rice Research Center,Changsha 410125,China;College of Bioscience and Biotechnology,Hunan Agricultural University,Changsha 410128,China;College of Agronomy Science,Hunan Agricultural University,Changsha 410128,China;Hunan Agricultural Biotechnology Research Institute,Changsha 410125,China)

机构地区：[1]湖南杂交水稻研究中心杂交水稻国家重点实验室,长沙410125 [2]湖南农业大学生物科学技术学院,长沙410128 [3]湖南农业大学农学院,长沙410128 [4]湖南省农业生物技术研究所,长沙410125

出　　处：《农业生物技术学报》2020年第12期2108-2117,共10页Journal of Agricultural Biotechnology

基　　金：湖南省自然科学基金(2019JJ40206);湖南杂交水稻研究中心科研创新基金(YB201907);湖南农业科技创新资金(2017XC09);国家转基因生物新品种培育科技重大专项(2016ZX08001004)。

摘　　要：叶色突变体是解析叶绿素生物合成机理的理想遗传材料。本研究通过辐射诱变从籼稻(Oryza sativa subsp.indica)品种’T98B’中获得了1份黄叶突变体(yellow leaf(t),yl(t)),分析了yl(t)的表型、生理特性及其突变机制。结果表明,yl(t)苗期叶片严重黄化,总叶绿素、叶绿素a和叶绿素b含量显著降低,分别为T98B的53.02%、55.35%和46.88%,但随着叶片发育成熟其叶绿素含量逐渐增加。遗传分析发现,yl(t)受1对隐性细胞核基因控制;利用yl(t)与正常叶色粳稻(O.sativa subsp.japonica)品种’日本晴’(Nipponbare,NPB)杂交后的F2群体将yl(t)精细定位在第6染色体1个42 kb的区域内;测序发现,定位区域内yl(t)的血红素加氧酶基因1(heme oxygenase gene 1,OsHO1)(GenBank No.LOCOs06g40080.1)的第2外显子与第2内含子之间的剪切位点"AG/GT"上缺失了"T",进一步经CAPS标记分析证实该位点与叶色表型存在高度相关性。利用3’端cDNA末端快速扩增技术(rapid amplification of cDNA ends,RACE)从yl(t)中获得了OsHO1的CDS,其在对应于野生型第2内含子的第20~22位碱基(TAA)处发生终止,导致全长CDS比野生型缩短了179 bp;经DNAMAN与CD-search分析推断,yl(t)的OsHO1编码产物C端较T98B缺失了59个氨基酸残基,可能降低血红素结合能力。实时荧光定量PCR分析发现,yl(t)中参与叶绿素合成途径的一些关键基因表达量发生了改变,OsHemA(hemin A gene)(编码谷氨酰-tRNA还原酶)上调而叶绿素酸酯a加氧酶基因(chlorophyllide a oxygenase 1 gene,OsCAO1)和OsNOL(non-yellow coloring 1-like gene)(编码叶绿素b还原酶)明显下调。本研究明确了yl(t)系OsHO1剪切位点变异,为深入认识OsHO1的生物学功能提供了遗传材料。Leaf color mutants are ideal genetic resources to reveal the mechanisms underlying chlorophyll biosynthesis.Here,a yellow leaf mutant yl(t),identified from indica rice(Oryza sativa)T98 B was analyzed on phenotype,physiology characteristics and mutation mechanism.The results showed that,the seedlings of yl(t)presented yellow leaves with a significant decrease of 53.02%,55.35%and 46.88%in the contents of total chlorophyll,chlorophyll a and chlorophyll b,respectively,compared with T98 B.When it grew to mature,the chlorophyll content increased.Further genetic analysis showed yl(t)was controlled by a nuclear recessive gene.By employing a F2 population from yl(t)crossed with the O.sativa subsp.japonica cv.Nipponbare(NPB)which presents normally green leaf,yl(t)was fine mapped in a 42 kb region on chromosome 6.Fragment sequencing confirmed that the heme oxygenase 1 gene OsHO1(LOC_Os06 g40080.1)would be the candidate of yl(t)that happened to mutate at the splicing site of exon2/intron2(AG/GT)with a deletion of’T’.Moreover,this point was proved to be associated with variation in leaf color by a CAPS marker’s verification.Later,by means of 3’rapid amplification of cDNA ends(RACE),the full-length CDS of OsHO1 in yl(t)was cloned.Compared with T98 B,the transcription of OsHO1 in yl(t)terminates at the 20~22 bp(TAA)on the second intron,thereby causing a reduction of 179 bp in the CDS sequence.Inferred by DNAMAN and CDsearch,59 amino acid residues at the C-terminus of OsHO1 in yl(t)would be missed to impair the ability of heme binding.In addition,qRT-PCR analysigbbbs found several key genes involving in chlorophyll biosynthesis expressed differently in yl(t).The OsHemA(hemin A gene)that encodes glutamyl-tRNA reductase was up-regulated,while OsCAO1(chlorophyllide a oxygenase 1 gene)and OsNOL(non-yellow coloring1-like gene)that encodes chlorophyll b reductase expressed lower.This study reveals yl(t)is an allele of OsHO1 to mutate at splicing site,and provides a new germplasm for further understanding of the biological func

关 键 词：水稻 黄叶突变体(yl(t)) 血红素加氧酶基因1(OsHO1) 叶绿素生物合成 剪切位点突变 

分 类 号：S33[农业科学—作物遗传育种]