摘要
水稻作为重要的粮食作物和理想的模式植物,其花发育调控的遗传和分子机制研究已成为当今分子生物学的研究热点。水稻的花器官雄蕊和雌蕊是保守的,许多实验结果均证实浆片是双子叶植物花瓣的同源器官,然而外稃和内稃与双子叶植物和其他单子叶植物的花萼器官的对应关系仍存在争议。水稻花发育突变体为研究相关基因表达与功能提供了理想材料,内外稃发育与稻米产量、品质关系密切,可见对水稻内外稃突变体进行研究具有理论和现实意义。
从水稻(Oryza sativa L)‘浙粳22’的6。CoY射线辐射诱变群体和‘秀水09’的EMS诱变群体中筛选获得4份月亮型颖壳突变体(moon rice glume,简称mrg)。对突变体mrg进行了表型观察、米质分析、基因的图位克隆和功能分析等,结果如下：
1月亮型颖壳突变体mrg形态结构特征：营养生长阶段,月亮型颖壳突变体mrg-1、mrg-2、mrg-3和mrg-4植株形态和野生型植株没有明显差别；突变体mrg浆片、雄蕊和雌蕊正常,但内稃和外稃弯曲,4个突变体表型变异程度略有差异。组织学分析表明,这种差异是由内外稃相互勾合不够紧密造成。
2月亮型颖壳突变体mrg稻米品质：稻米外观品质方面,月亮型颖壳突变体粒长和粒宽均较野生型小,长宽比增加,千粒重、垩白率和垩白度降低；稻米加工品质方面,月亮型颖壳突变体稻米的糙米率、精米率和整精米率均较野生型低；稻米营养品质方面,月亮型颖壳突变体蛋白含量较野生型高,为9.917mg/g；稻米蒸煮食味品质方面,月亮型颖壳突变体胶稠度较野生型高,为83.06mm,属于软胶稠度；直链淀粉含量和碱消值较野生型减小,分别为13.18%和7.02,属于低糊化温度,月亮型颖壳突变体的蒸煮品质发生变化。
3突变性状的遗传分析：突变体mrg-1和mrg-2分别与粳稻‘浙粳22’杂交,F1表型与野生型一致,说明mrg为隐性突变导致。根据F2表型及χ2测验结果表明,正常株与突变株的比例符合1对基因控制的分离比3：1,即该突变性状是受一对隐性基因控制。
4 MRG基因的精细定位和候选基因的克隆分析和确定：利用图位克隆方法,将MRG基因定位于2号染色体InDel标记J59与J95之间,两者相距194Kb,该区域位于AP004081、AP005303和AP005691三个BAC所构成的重叠群上。通过测序得知mrg-1、mrg-2、mrg-3和mrg-4的Os02g0811000基因存在不同形式的突变。根据测序结果,在突变位点存在酶切位点差异。酶切检测突变群体,此突变位点与突变性状完全连锁,确定了Os02g0811000就是MRG的候选基因。
5 MRG蛋白分析：MRG基因没有内含子,全长cDNA序列(Genbank登录号：NM_001055008.1)为1587bp,其中ORF区为747bp,5'-UTR区为503bp,3'-UTR区为337bp。MRG基因的编码蛋白(Genbank登录号：BAF10387.1或NP 001048473.1)为248个氨基酸,第79-210氨基酸构成DUF640结构域,MRG蛋白的分子量为25.89kD,等电点(PI)为9.62。MRG蛋白具有核定位信号(KKKR,氨基酸205-208),MRG-YFP融合蛋白亚细胞定位结果显示,MRG蛋白定位在细胞核。MRG蛋白含未知功能DUF640结构域,通过对含该结构域的同源蛋白比对分析,发现MRG蛋白与已报道的G1和LSH1属于不同的分支。DUF640结构域具有保守的氨基酸序列,突变体mrg-2和mrg-3的MRG基因发生单碱基突变的位点都位于该结构域保守位点。
Rice is an important food crop and model plant. The genetic and molecular mechanism underlying the regulation of floral development has become one of important subjects. There is evident homology for stamens and carpels between monocots and eudicots. Lodicules are interpreted as homologous to a single perianth whorl of other monocots. Considerable debates exist in the relationship beteen palea and lemma structures and dicot sepals. The mutant of floral development is important for the studies on gene expression and function. The development of palea and lemma structures is directively related to rice quality and productivity. Research on rice reproductive mechanism is significant both in theory and practice.
Four moon rice glume (mrg) mutants were obtained by screening the Zhejing22 mutant pool which was theated with 60Co y-ray and Xiushui09 mutant pool which was theated with 0.8% EMS. In this study, we have made the investigation of the floral organs, rice quality and the characterization of a novel gene named MRG. The main results are the following:
1 The investigation of floral morphogenesis:No abnormalities were observed in the vegetative stage. In the ripening stage, we found the lodicules, stamens and carpels of mrg were nomal, but the paleas and lemmas of mrg were twisted and degenerated, however, the phenotypic severity differed significantly among the four mutants. According to histological analysis, the difference was caused by the loose joints of palea and lemma.
2 The rice quality of mrg: We analyzed the rice quality of mrg and wild-type. The impact of palea and lemma development on grain shape was larger than other qualities. The grain length and width of mrg was much shorter than that of wild-type, resulted in the increase of length width ratio and decrease of thousand grains weight. The chalkiness grain rate and degree of white core of mrg decreased. The percent of protein content increased compared with wild-type, which was 9.917mg/g. cooking and tasting quality of mrg was different for the decreace of amylose content and gel consistence.
3 Genetic analysis:mrg-1 and mrg-2 mutants were crossed with Zhejing22 respectively. The F1 population of two crosses showed wild-type phenotype. Investigation for F2 population showed that the segregation of normal plants and mutant plants fited a ratio of 3:1. The result of genetic analysis indicated that the traits of mrg was controlled by a single reeessive gene.
4 Fine mapping of MRG, determining and cloning analysis of candidate genes: The gene MRG was mapped between InDel maker J59 and J95 on chromosome 2 with physical distance of 194 Kb by a map-based clone stratege. In this region, there are three BACs named AP004081, AP005303 and AP005691. We sequenced the cDNA and DNA of Os02g0811000 in mrg mutants and wild type, and found mutation in the open reading frame region of Os02g0811000 gene. New restriction sites were born for the mutation of Os02g0811000 gene in mutants, all of the mutant plant population was digested. These results indicated that Os02g0811000 gene was the MRG gene.
5 Functional Analysis of the MRG:The 3'non-coding region of the MRG transcript is 197 nt long and the 5'non-coding region is 49 nt long. The ORF of MRG contains no introns. From the cDNA sequence we obtained, it was predicted that MRG encodes a protein of 248 amino acids which has a conserved domain named DUF640. The MRG protein contains putative nuclear localization signal (KKKR, amino-acid residues 205-208). MRG protein was localized at the nucleus using YFP localization. Phylogenic tree of proteins containing a DUF640 domain indicates MRG belonged to a new branch that is different from G1 and LSH1. the mutated sites in mrg2 and mrg3 are in the conserved region of DUF640.
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