Screening The T-DNA-inserted Rice Mutant Pool And Cloning A Functional Gene, DH1, Which Is Required For The Formation Of The Floral Glumes In Rice

T-DNA tagging is a high throughput method for identifying and cloning novel genes and has been widely applied in functional genomics research in dicotyls, but its application in monocot is still in original and exploring stage. In this dissertation, by screening 4416 T-DNA tagged lines, we analysed the mutation type and mutation frequency of different traits, and discussed the screening procedure and strategy for T-DNA inserted mutagenesis, isolation of T-DNA flanking sequence and integration characteristics of T-DNA. Furthermore, for a degenerated hull mutant, we cloned and validated its corresponding functional gene, DH1, and assayed its expression pattern. For a multi-glume mutant, we made a morphogenesis, anatomical observation and primary genetic analyses. The main results can be described as follows.i. By screening 4416 rice T₁ tagged lines from transformants of two parents, ZH11 and ZH15, we found many lines showed obvious morphological mutations, which could be divided into two types, fake-homozygous mutation and separating mutation in a tagged line. The mutation phenotype was related to 14 kinds of traits such as plant height, heading date, leaf shape, leaf color, tiller capability and degree of tightness, panicle shape, spikelet number, grain shape, disease-like mutation, male sterility, awn, and so on. Among them, plant height, heading date, leaf color and male sterile had a comparatively high mutation frequency (over 1%). The mutation frequency of plant height and leaf color had no significant change between different years or transformed parents, but the frequency of heading date and male sterility varied greatly, suggesting that environment had a great effect on the expression of latter two traits. By conducting continuously co-segregating analyses in T₁ and T₂ generation, three T-DNA-inserted mutants with misshapen panicle or spikelets were identified, and the morphological mutation frequency from T-DNA insertion was about 1.37‰, which would provide a base for cloning correlative functional genes.We selected randomly 42 lines with mutation phenotype and obtained 40 flanking sequences from 39 of the tagged lines by plasmid rescue or TAIL-PCR, of which, 26 were vector backbone sequence and 14 had good identity to rice genome sequence. The blastn result showed the T-DNA was preferentially integrated into protein-coding region in plants.In this study, we established two methods for isolating the T-DNA flanking sequence, plasmid rescue and TAIL-PCR. The former had the advantages of higher rate in obtaining positive clones carried with T-DNA flanking sequence, higher efficiency in sequencing, and longer length in isolated flanking fragments for a certain tagged line, but it required the higher quality of template DNA, and its manipulation procedure was laborious. The latter had a higher efficiency in isolating flanking sequences on a large scale and constructing data pool of flanking sequences, but it appeared imperfect on lower rate in obtaining specific PCR product for a certain line, limited length of isolated flanking sequence, and so on. Combining these two methods for the detection of a specific tagged line, we would enhance the odds of obtaining flanking sequence.