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Positional Cloning and Functional Study of the Rice Gene Eui Controlling Elongation of Uppermost Internode

Author: XuYongHan
Tutor: SunZongXiu；HeZuHua
School: Zhejiang University
Course: Genetics
Keywords: rico(Oryza sativa L.) eui mutant Eui gene mapping-based cloning TAC (transformation-competent artificial chromosome)vector GA(Gibberellin) functional complementation.
CLC: S511
Type: PhD thesis
Year: 2004
Downloads: 672
Quote: 1
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Abstract


In this experiment, systematical research was carried out on a recessive rice mutant eui. The eui mutant possessed the mutated traits including the remarkably rapid elongation of the uppermost internode at heading stage as to produce a near-dubling in length of the uppermost internode, great panicle exsertion and an increase of 12% in panicle length, thus leading to the noticeable extension of whole plant. Here author presented the main research results as follows:(1) By crossing a pair of NTLs 307T(mutant, euieui, ftomjaponica variety eui stock) and Zhengshan 97(wild type, EuiEui), a mapping F2 population of about 6000 individual was constructed to isolate the Eui gene;(2) According to DNA sequences of chromosome 5 of Nipponbare available in http://140.109.57.19/aspgc/sequencing.php and 93}\(indica)in http://btn.genomics.org.cn/rice/. more than 70 pairs of primers for CAPS makers were designed in a 14.5 cM region of the long arm of chromosome 5. Among them, some were directly adopted from the rice genomic program (http://rgp.dna.affrc.go.jp/publicdata/) that showed polymorphism between Nipponbare (japonica) and Kasalath (indica), and most of the CAPS primers were newly designed based on single nucleotide polymorphism (SNP) between japonica (Nipponbare) and indica (9311) genomes. PCR products of most of primers showed polymorphism between 307T and Zhenshan 97. In cases of non-polymo rphism, we cloned and sequenced the PCR products to find the special SNP between 307T and Zhenshan 97for new CAPS markers. Thus, total 45 CAPS markers were identified between 307T and Zhenshan 97 by at least one of the 26 restriction endonucleases;(3) By using a few dominant plants, 198 F2 recessive plants and some CAPS markers, linkage analysis was carried out and the result revealed that Eui gene was located in a 6cM intervalin the middle area of the long arm of chromosome 5;(4) In the further mapping of the Eui gene, using 13 newly-developed CAPS markers, morethan 1500 homozygous recessive plants (euieui) and a few dominant plants of the F2 population, we at last constructed the high-resolution genetic map and BAG contig of Eui gene locus, in which the Eui locus was narrowed down to a DNA region of 0.6 cM with the length of 24kb;(5) In the 24kb target gene region, 2 ORFs were predicated by the software GENESCAN and confirmed by public cDNA(EST) mining;(6) By Sequencing and comparing the whole target gene DNA region of 24kb in both mutant and wildtype plant, the probable mutation sites were spotted that would accordingly indicate the predicated Eui candidate gene;(7) Functional complementation experiment: transform the mutant plants (including eui stock and 307T ) with the TAC expressing vector containing the whole target gene DNA region of 24kb(the whole insert was 40kb).Some lines (TO and Tl) reverted to the wildtype phenotype;(8) The identification of transgenic plants. PCR and Southern blotting analysis showed that the target DNA fragment had been integrated into the rice genomic DNA and was inherited stably in the second generations(T2) of most of transgenic plants. However, in the progeny(including Tl and T2) of transgenic plants(TO), the ratio of the eui plants to Eui plants seemed not to fit the theoretical 1:3 distribution as predicted by mendelian inheritance;(9) The contents of BRs and GAs in the uppermost interaode of both eui mutant (307T) and wildtype (308D) plant was measured and the results revealed that, there was no obvious difference between the two plants for the endogenous BRs and sterols level, however the mutant (307T) contained the remarkably elevated GA1 and GA4 (the major bioactive GA in rice vegetative tissues) and some precursor GAs, a clue that the eui trait would be well caused by the striking increase of bioactive GAs;(10) At seedling stage, 307T and 308D were treated with various concentration of GA, and then it was found that 307T showed higher sensitivity to the exogenous GA;(11) Cell biological study on eui mutant: 307T and 308D were gro

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