| Plant male-sterile mutants are ideal materials for research on gene expression and genetic engineering. The phenotypic characteristics of them consist of many forms:the shrinking, distortion or disappearance of male organs; abnormal microspores formation leading to no pollen in anther; abnormal microspores leading to abortive pollen; normal pollen formation but anther indehiscence; pollen cannot be recognized by and germinate on stigma, etc. Male sterility in plants was caused by a variety of reasons. A close relationship between plant hormones and male sterility were found by identification of kinds of mutants, such as mutants with pollen development defects and anther indehiscence, most of which correlated with jasmonic acid and auxin. In this study, we mainly focus on the relationship between rice anther indehiscence and auxin, so the gene was named ANTHER INDEHISCENCE2(AID2). Indole-3-acetic acid (IAA) is the main form of auxin in plant, it is involved in almost all aspects of plant growth and development, including cell division, cell elongation, cell differentiation, plant tropism, floral organs development and formation of the vascular system. The synthesis of auxin mainly arises in vigorous growth organs, such as coleoptile, the young root tip, shoot apex, leaves and immature seeds. When different developmental process requires auxin to play a role, it is transported to various parts of the plant mostly in the way of polar transport. However, the accumulation of high auxin level will adversely affect plant growth and development. Numerous abnormal phenotypes are induced by overproduced auxin, in which male sterility is one aspect.Asian cultivated rice(Oryza sativa L.) and African cultivated rice(Oryza glaberrima Steud.) are the two major cultivated rice species in the world. They originated from different wild ancestors, but both have the AA-genome (2n=24) and similar sequence arrangement. Studies have shown that hybrids of them exhibited transgressive heterosis for dry weight and tiller numbers per plant. In addition, African cultivated rice harbors various types of disease and insect resistance, drought tolerance and saline-alkaline resistance genes. However, serious hybrid sterility blocks the application of heterosis and useful genes. With the large scale extension of several high-yielding varieties, the rice genetic basis has become increasingly narrow, which seriously affected the increase of rice yield. Therefore, to broaden the genetic basis of rice, induction of useful genes from near relatives is considered to be one of the most important means to break the current breeding bottleneck. So, the excavation, mapping and cloning of rice hybrid sterility genes have important theoretical value and practical significance to understand and finally overcome hybrid sterility.This study was divided into two aspects. Firstly, we obtained a rice male-sterile mutant by radiation mutagenesis in rice japonica variety "Nipponbare". We carried out a detailed cytological analysis of the mutant and finally separated the gene through positional cloning method. A primary functional analysis of the gene was also performed. Secondly, we constructed a near-isogenic line for rice inter-specific hybrid pollen sterility gene S19, and fine mapped the S19gene. Meanwhile, cytological observation of hybrid pollen development process was also performed.The main results of the paper were as follows:1. By morphological and cellular observation, we found that the main features of the mutant were as follows:no obvious difference was found in plant height, tiller number and heading date between wile-type and mutant. Floral organ was normal in mutant. At anthesis, however, the lemma and palea could not open and the anther appeared indehiscence. By consecutive semi-thin section of anthers at different development stages, we observed that the anther wall developed normally with tapetum and middle layer degradated on schedule. But the four locules remained separated with each other by anther dehiscence stage, unable to complete the dehiscence. Pollen grains of the mutant could be deeply stained by iodine-potassium iodine (I2-KI). Three nucleuses were clearly seen in mature pollen grains by DAPI staining. Using electron microscopy and transmission electron microscopy, we observed normal pollen wall, germinating pore and starch granules of the mutant pollen. Pollen germination in vitro was not successful. By artificially breaking the mutant anther to release pollen grains to its own stigma, no signs of pollen germination observed either, which indicated that although the pollen developed normally, they did not obtain germination ability. When the mutant plants were pollinated with wild-type pollen, they restored seed-setting. So the mutant could be regarded as a male sterile mutant. 2. Genetic analysis of the offspring of mutant/Nipponbare heterozygous plants showed that normal and mutant plants segregated in a3:1ratio, indicating monofactorial recessive inheritance of the mutant characteristic.2100mutant plants segregated form a total of18000mutant/Dular F2population. Finally the gene was mapped to a52kb fragment on rice chromosome4through SSR and InDel markers analysis. By sequencing the52kb region and comparing with the published Nipponbare sequence, a single nucleotide C deletion was found at the24position from the start codon of one of the genes, resulting in premature translational termination. The wild-type gene was predicted to encode300amino acids while the mutant gene encoding only48amino acids. So it was chosen as the candidate gene for transgenic verification. A total length of6083bp DNA fragment from wild-type including the whole gene region,3.2kb upstream from the start codon and1.6kb downstream from the stop codon was cloned into the binary vector pCAMBIA1305.1and transformed into the mutant callus. The obtained transgenic positive plants were found to be complemented by the wild-type gene. To further confirm the candidate gene, we also constructed an RNAi construct containing a272bp specific fragment from the first exon and transformed it into the wild-type background. Most transgenic positive RNAi plants displayed varying degrees of mutant phenotypes. Thus the candidate gene was ultimately affirmed.3. It is considered that male sterility and plant hormones are closely linked. Panicles of wild-type sprayed with different kinds and levels of plant hormones, and it was found that panicles treated with10-2M IAA and10-4M2,4-D displayed no flower opening after24h. But they restored flower opening after72h, which could be concluded that high levels of auxin indeed delayed rice flower opening. The free IAA levels in both anther and ovary of wild-type and mutant were determined. IAA content in mutant was3to4times higher than wild-type. The rice auxin biosynthesis key gene OsYUCCAl was overexpressed in wild-type background, which caused phenotypes as flower not opening, anther indehiscence. These results further validated that the male sterility in rice here was induced by auxin and the gene identified here might be related to auxin metabolism.4. Results of real-time fluorescence quantitative PCR and promoter-GUS transgenic rice plants indicated that the gene expressed in various parts of plant, but the highest expression was found in anthers at late development stage and fertilized ovaries. The peptide containing143-292amino acids was used as an antigen for polyclonal antibody. Western-blot analysis of anther protein extracts observed a clear band at the position of molecular mass of target protein in wild-type, while the mutant did not show any hybridization signal, indicating that the functional protein was absent in the mutant.5. The14C-sucrose isotope tracer experiment indicated that radiations in mutant anther and palea/lemma were lower than wild-type. However, higher radiation was detected in the three internodes of mutant stem. When the wild-type spikelets were pretreated with IAA, the transport efficiency of sucrose decreased to that of mutant, indicating that the high level of auxin inhibited sucrose transporting to floral organs. The measured sucrose and glucose levels in mutant anther were lower than wild-type. The sucrose and glucose concentrations in mutant lodicules were significantly lower than wild-type, and sucrose consisted of the predominant soluble sugar in lodicule. The osmotic pressure was also lower in mutant lodicules. Since sucrose was recognized as an osmotic pressure regulator, we inferred that the failure of flower opening in mutant was caused by low osmotic pressure in lodicules, which might resulting from sucrose transport barrier in mutant plants.6. By using of near-isogenic lines derived from an initial cross between ajaponica variety Dianjingyou1(recurrent parent) and an African rice variety (donor parent), a hybrid polle sterility gene S19was identified and fine mapped on rice chromosome3by12,870F2plants from the hybrids of the near-isogenic line and Dianjingyou1. The typical pollen semi-sterility was observed in F1hybrids. Molecular analysis revealed that the semi-sterility was caused by the abortion of most male gametophytes carrying the S19allele from the japonica variety Dianjingyou1. Cytological study of pollen developmental stages indicated that pollen abortion occurred at the late binucleate stage because of a starch accumulation obstacle in some pollen grains. The pollen wall of hybrids developed normally. |
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