Cloning And Verification Of Flowering-related Gene In Ccri36

Short-season cotton, an important resource of cotton varieties, plays a vital role in the contradiction settlement between large amount of people and small area of farmland as well as the conflict of acreage between grain and cotton, and optimizing agricultural structure. Specially, in development situation of the country to ensure grain production, the acreage of short-season cotton will be extended. In the future, by use of the combination agricultural biotechnology with traditional breeding, together with introduction and utilization of cotton high yield and high quality germplasm resources, further improvement the earliness and productivity of short-season cotton and coordination the contradictions between early maturity and high yield, early maturity and quality are the main developmental direction of short-season cotton breeding and production.CCRI36 is a short-season variety of upland cotton, with 107 days growth period. In the research CCRI36 was used as the material, gene expression profile of main stem growing point was analysed by Arabidopsis thaliana Affymetrix genome-wide chip during the first true leaf expansion and the second true leaf expansion of CCRI36, and 3 genes related to flower bud differentiation were screened and cloned, and conducted spatial and temporal expression pattern analysis and transgenic functional verification, and obtained a portion of precocious transgenic plant, thus it provides significant theoretical implications and applicable value for the promotion of cotton flower developmental process and the acceleration of genetic improvement of cotton early-ripening traits. The experiment results and conclusions are as follows:1. Through microscopic examination of main stem growing point of precocious variety CCRI36 and late-maturing variety TM-1, it turns out CCRI36 starts flower bud differentiation at the time point of two true leaves expansion, whereas TM-1 do so during three leaves expansion. This indicates flower bud differentiation of precocious variety is earlier than that of late-maturing variety.2. Through Arabidopsis thaliana Affymetrix genome-wide chip, gene expression profile of main stem growing point is analysed during the first true leaf expansion and the second true leaf expansion of CCRI36, 360 significantly differential expressed transcripts are screened from 22,700 transcripts, among which the expression of 210 transcripts were up-regulated and 155 transcripts down-regulated.3. Functional classification is carried out against 365 differentially expressed genes by using MIPS database, and this mainly contains transcriptional factor, putative regulatory protein etc., and 99 unknown function or new genes. Moreover, 365 differentially expressed genes are conducted blastn analysis in website "http://compbio.dfci.harvard.edu/tgi/cgi-bi n/tgi/gimain.pl?gudb=cotton".4. 10 ESTs are selected to conduct RT-PCR analysis from cotton EST corresponding to 360 significantly differential expressed Arabidopsis thaliana probe, the result indicates that RT-PCR result is exactly consistent with microarray data analysis. 2 transcripts are selected to carry out QRT-PCR analysis from 10 differential expressed transcripts, and it turns out RT-PCR, QRT-PCR and microarray data analysis are in agreement with each other completely, showing that it is absolutely viable to dissect changes in gene expression during cotton flower bud differentiation by Arabidopsis thaliana genome-wide chip.5. A transcript highly homologous to Arabidopsis thaliana VRN1 gene is screened from 155 down-regulated transcripts, and QRT-PCR of different part reveals that it expresses in main stem growing point, root, petal, stamen, ovule all, especially espresses dominantly in main stem growing point; QRT-PCR analysis of different phase of main stem growing point is carried out, and the result indicates that it up regulates highly during first true leaf expansion, whilst during two true leaves expansion(flower bud differentiation) its expression is down-regulated slightly, thereby speculating it might be associated with flower bud differentiation of main stem growing point. In the experiment we've cloned its full length, and registered in NCBI as GU929695, and denoted as GhV1 gene.6. In this study we utilize CCRI36 full-length normalized cDNA library constructed by our lab, and have screened two genes GhCO, GhTM6 related to flower development, and analysed expression pattern of various part and developmental stage by using QRT-PCR. The results show that GhCO gene is highly up-regulated in flower and stem, GhTM6 highly up-regulated in flower, stamen, pistil etc. We hypothesize that they might involve in flower development process.7. Through QRT-PCR analysis of GhV1,GhCO,GhTM6 under different lighting conditions and temperature, we realize that very low is the expression level of three genes of GhV1, GhCO, GhTM6, after 12h low-temperature treatment the expression level rises up sharply, after 36h declines gradually. GhV1 gene takes on rhythmic alterations under conditions of long day(16 h light/8 h dark), short day(8 h light/16 h dark)and middle day(10 h light/14 h dark). GhTM6 and GhCO are insensitive to sunlight under short-day treatment.8. By means of Agrobacterium-mediated and pollen tube injection methods three genes of GhV1, GhCO, GhTM6 are conducted tobacco and cotton genetic transformation. The result implies the expression of exogenous GhV1 gene brings about changes in tobacco's stamen, and there are 3 long stamens and 2 short ones in transgenic plant, and the flowering time is somewhat earlier than wild type. The transgenic plant of GhCO gene presents variation of 4 petals and 4 stamens, and wild type plant starts flowering with 19 leaves, whereas transgenic plant begins flowering with 14 leaves. In addition, the expression of exogenous GhCO gene results in dwarf of transgenic plant. Transgenic plant of GhTM6 appears mutation, with 6 petals and 6 stamens, and wild type plant begins flowering with 19 leaves, whilst transgenic plant starts flowering with 18 leaves. The expression of exogenous GhTM6 gene leads to internode elongation and height increase of transgenic plant. By way of Agrobacterium- mediated and pollen tube injection approaches transgenic plants of GhCO, GhTM6, GhV1 have already acquired, and the results remain further observation.