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Map-based Cloning and Functional Analysis of Tillering Gene ext-M1B in Rice

Author: LiangYueYang
Tutor: LiPing
School: Sichuan Agricultural University
Course: Crop Genetics and Breeding
Keywords: rice ext-M1B gene tillering F-box ubiquitination
CLC: S511
Type: Master's thesis
Year: 2010
Downloads: 152
Quote: 1
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Abstract

Rice is an important food crop in the world. Improving rice yields is the common mission of breeder and biotechnology researchers. Tillering is a basic characteristic in rice, which is determined grain yield. Therefore, research on rice tillering has both important theoretical significance and application value. With the swift development of genetics and experiment methods in molecular biology, many genes involved in the rice tillering had been cloned that provided theory foundation to further elucidate the complex molecular mechanism.The excessive tillering mutant named M1B- ext, was found in the F2 generation in a cross of M1B and GMS-1(a genetic male sterile).F2 and F1 plants of three reciprocal cross (2480B、D62B、G46B、G683B、M1B) was used to genetic analysis. The results showed that all the F1 generation expressed normal tillering. The character segregation was found in the F2 generation, normal type and excessive tillering type were segregated at mendelian ratio of 3:1 by x2 test. It is realized that the mutant trait of ext-M1B is controlled by one recessive nuclear gene.By linkage analysis of molecular markers the ext-M1B gene was located on the short arm of chromosome 6 between SSR markers RM19398 and RM510 in a 51.8 kb region. We found that a coding F-box protein contained LRR repeat amino acid sequence in Bioinformatics, and highly homologous to MAX2 which is inhibition of branching in Arabidopsis, is likely to be candidate gene (named OsMAX2). The DNA sequencing results also showed that the region of mutant base sequence at sites of 1012 had been changed from GAT to GCCT. Amino acid sequence alignment suggest that it may cause the frameshift mutation, as a result, the 372 amino acid changed into a stop codon TAA,so thus,the OsMAX2 gene maybe the ext-M1B. Real-time quantitative fluorescent PCR analysis was used in this study for expression of transcript levels in roots, stems and leaves. It indicated that the highest transcription level were observed in leaves,stem expression was slightly higher than the root, we speculated that the ext-M1B genes may be occurred in various tissues involved with a removable substance.Recent research has shown that the MAX2 may be involved in the pathway of shoot branching inhibition via strigolactones.To investigate if ext-M1B acts in the strigolactone pathway, we had analyzed D10,D14,HTD1 and D27 transcript levels in wild-type and M1B-ext mutant. The results showed that D10, HTD1 and D27 expression levels was down-regulation in MIB- ext mutant, but the expression of D14 was significantly higher than wild type. The results showed that the ext-M1B gene was likely involved in the strigolactone pathway.To clearly define the OsMAX2 gene function, the coding region fragments were amplified by PCR with the primers designed according to this gene sequences. Then we had constructed the over and interference expression vector. The transgenic plants were obtained by agrobacterium-mediated transformation of plant transgenic technology. Now the functional verification test is ongoing.In this study, genetic analysis of a rice excessive tillering mutant MIB- ext showed that the mutant trait was controlled by a single recessive gene (ext-M1B), and the OsMAX2 maybe the candidate gene. We supposed that ext-M1B may be involved in the shoot branching inhibition via strigolactones pathway as a signaling receptor. The strigolactones signaling can enhance the interaction and identification between ext-M1B/F-box E3 ubiquitin ligase complex and target protein that is an inhibition of the hormone transcription factors. After the ubiquitination modifications by E3 complex, the transcription inhibitor was degradation into 26s proteasome. As a result, the inhibiting effect was relieved, and the gene relate with strigolactones begin to transcribe.

Map-based Cloning and Functional Analysis of Tillering Gene ext-M1B in Rice

Abstract

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