【作者】 阮伯胜；

【导师】 石春海；

【作者基本信息】 浙江大学 ， 作物遗传育种， 2008， 硕士

【摘要】 突变体是进行功能基因组学研究的重要材料,许多水稻基因现已得到克隆。本研究利用化学诱变剂甲基磺酸乙酯（EMS）进行粳稻品种日本晴“Nipponbare”的突变体诱变。利用实验室水培方式,在40,162个M₂分离群体中筛选得到981个苗期突变体（突变频率为2.44%）,其中以白化突变为最丰富（突变频率为1.3%）,其次为矮化苗和卷叶突变,突变频率分别为0.24和0.18%。在根系突变体中,共得到117个根突变如长根、短根、无不定根、少侧根、异常根等不同类型的根突变体,其中以长根变异频率为最高;通过M₃鉴定,其中41个根突变体是可遗传的。苗期性状筛选后,利用0.25%Batas、1.2%NaCl和0.25%PEG6000进行抗除草剂、耐盐和耐旱突变体筛选;在M₂中发现53个抗除草剂突变体、42个耐盐突变体和33个耐旱突变体,其突变频率分别为0.43、0.32和0.22%。经过验证在M₃有29个抗除草剂突变体（株系）、17个耐盐突变体和13个耐旱（株系）表现是纯合体,其它突变对抗性有分离现象。在大田中,2006年利用M₂进行突变体筛选的结果表明,地上的植株性状变异最为丰富,占突变体库的56.15%。其中以茎秆变异的突变频率为最高（37.68%）,其次是叶片变异、穗部变异和籽粒变异（突变频率分别为33.50%、16.75%和12.07%）。其它一些突变体如根系、抗性、生理变异等为43.85%。同时还发现了一些目前尚未报道的双胚苗和白穗等人工诱变产生的突变体。2007年利用M₃进行上述突变体的重复鉴定,获得了412个变异性状能够稳定遗传的突变体,其中叶片、茎秆、穗部和籽粒性状等地上植株性状的突变体占突变体库的59.95%。对4,043份M₃糙米材料进行了稻米品质性状和品质性状的突变体筛选。结果筛选到116份糙米外观品质性状的突变体（如垩白大、大胚、大米粒、小米粒、短圆米粒、长形米粒、糯性米、和红米等）。同时,利用近红外反射光谱技术,筛选到435份直链淀粉含量、胶稠度、碱消值、蛋白质含量以及氨基酸的突变体。通过对M₄糙米外观品质性状的表型重复鉴定,发现75份外观品质性状的突变体能够稳定遗传。在黄化、卷叶、抗除草剂、长根突变体变异性状的遗传分析中,发现这些性状表现为盂德尔遗传,符合3:1分离比,属于单基因突变,遗传模式简单,有利于今后突变基因的精细定位及分离克隆。利用粳稻日本晴的黄化突变体与籼稻93-11的黄化突变体杂交,进行黄化突变基因的等位性测定,发现绿色苗与黄色苗的分离比符合9:7分离比,即日本晴的黄化突变基因和93-11黄化突变基因是非等位,而且是互补关系。更多还原

【Abstract】 Now-a-days, rice is markedly diversified from the view-point of genetics, rice mutant has become prerequisite for morphology, physiology as well as functional genomics research, and many genes have been isolated from different mutants in rice. In present study, the seeds of japonica variety （Oryza sativa L. ssp. japonica） Nipponbare were treated by ethyl methane sulphonate （EMS） in order to construct the mutant library. From 40,162 M₂ seedlings, total of 981 mutants were screened with the mutant-induced frequency of 2.44%, in which the albino was highest frequency （1.3%）, followed by the dwarf and rolled leaf （0.24% and 0.18%, respectively）. By using hydroponics method, whole root system could be observed clearly for screening root mutants. About 117 root mutants comprising of long root, short root, less adventitious root, less lateral root and abnormal root was identified; but only 41 root mutants were found stable in inheritance after M3 validation. After morphological mutants were picked up, the remaining seedlings were sprayed with 0.25% Basta （herbicides） solution or treated with 1.2% NaCl solution or placed in 25% PEG 6000 solution for testing ability of herbicide resistance, salt tolerance and drought tolerance respectively. Fifty three herbicide resistance mutants, 42 salt tolerance mutants and 33 drought tolerance ones were identified with frequency of 0.43, 0.32 and 0.22 respectively. By inspection in M3, only 29 herbicide resistance mutants, 17 salt tolerance mutants and 13 drought tolerance ones could propagate to their offsprings without segregation.In 2006, mutants of morphological traits, after screening in M₁ and transfer to the field were found to occupy 56.15% of mutant library in M₂. Among them, culm and tillering trait mutant was most abundant with frequency of 37.68%, followed by leaf, panicle and spikelet mutants with frequency of 33.5%, 16.75% and 12.07%, respectively. Mutants for root traits, abiotic stress resistance and physiological traits occupied the remaining 43.85%. Besides, present study also identified some special mutants such as polyembryonic mutants and white panicle mutants also were captured, albeit with low frequency and could be propagated stably to next generation. M3 validation carried out in 2007 revealed that different mutants, including leaf, root, plant height, culm, panicle, spikelet, fertility and heading time could be stably inherited from generation to generation. By selecting the typical and stable mutants, a library of 412 EMS-mutagenized mutants, which were mostly stable in inheritance, was constructed in M3. And mutants of morphological traits （consisting of leaf, culm and tillering, panicle and spikelet traits） still occupied highest frequency of 59.95% of mutant library.From 4,043 brown rice materials, a pool of grain quality mutants （including brown rice appearance and rice quality mutants） was conducted. 116 mutants for brown rice appearance （including large chalkiness, big embryo, broad grain, small grain, round shaped grain, slender, glutinous rice, red colored rice）. Near infrared spectroscopy （NIRS）, which has several well-known advantages, was used for screening rice quality mutants and 435 nutrient characteristics （including amylose content, gel consistency, alkali spread value, protein content and amino acids） were also classified in M3. Validation in M4 showed that about 75 mutants brown rice appearance which were screened in M₃ were stable in inheritance （homozygous）.Some typical mutants （such as green yellow, rolled leaf, herbicide resistance, long root） were chosen to make hybridization with wild type. Genetic analysis results showed that, segregation in F₂ of these mutants tallied with 3:1 ratio of Mendelian inheritance for single gene controlled traits. By making an intesubspecific crossing between green yellow mutant of Nipponbare and green yellow mutant of 93-11, segregation in F₂ which suited ratio of 9:7, revealed that two mutants were controlled by two non-allelic genes that interact complementarily. These informations might be essential to elucidate the biological function of rice genes and possibly facilitate genetic analysis and positional cloning.更多还原