Construction Of CDNA Library And Cloning Of Development Related Genes Of Young Buds In Restorer Line Cotton

Cotton is an important economic crop in our country. How to use cotton heterosis efficiently has become one of the world's cotton breeding focuses. Three-line hybrid cotton is an effective way for cultivating the cotton hybrids and using heterosis, which will create huge economic and social benefits. However, due to limited resources of restorer line and few of superior three-line cotton, if, restore related genes would been cloned from cotton by genetic engineering technology and further work is progressed, it is extremely significant to enrich restorer resources and application of three-line hybrids.In this study, insect resistant three-line hybrids restorer line was used to be the material, which is carrying the fertility restoration gene from G.harknessii and the fertility enhancer gene from upland cotton. cDNA library of restorer line young bud was constructed and diversity gene of male-sterile line and fertility line was screened. The result as follows:1. Using restorer line 3⁵ day's young bud as material, RNA extraction efficiencies were investigated by using hot borate /protease K method and Trizol method respectively. The result indicated that RNA extracted by hot borate method showed better quality and integrity. Cotton gossypol browning phenomenon has been restrained effectively.2. Purification and analysis results of mRNA revealed extracted mRNA accorded with quality requirement for cDNA library construction. Cotton restorer line young bud cDNA library was constructed. Unamplified library contained about 1.6×10⁶ clones. The recombinant rate of the library was 92% and the average length of the cDNA library was 1.9kb. The titer of the amplified library was about 7.7×10⁹ pfu/ml, which can be applicable to gene expression and cloning study. This work has established the good basic for future research.3. According to F₂ restorer line SSH library, an unknown functional EST sequence was been cloned. A pair of special primers was designed according to it. A positive clone was screened by using PCR from cotton restorer line cDNA library. Analysis of positive clone and conclusions as follows:(1) The full-length of the positive clone is 1426bp. This nucleotide fragment was analyzed by using EST data in NCBI. The sequence showed 98% similarity to cDNA clone from fiber SSH library of Gossypium hirsutum, and showed 99% similarity to the sequence of Oryza sativa; it also showed high similarity to 1 day's fiber after anthesis Gossypium hirsutum cDNA, 3 weeks after planting Gossypium hirsutum cDNA and Arabidopsis cDNA. However, the functions of these cDNA fragments haven't been verified.(2) An entire ORF is from 135～1343bp which the full-length of 1209bp, which has an initiation codon of ATG, a termination codon of TGA and encodes a deduced amino acid sequence of 402 residues.(3) Analysis of the amino acid sequence illuminated the protein encoded by this gene is similar 98% with IS10 transposase in Lycopersicon esculentum. This gene was named GHtp1209.We draw a conclusion that, this gene may affect the development of cotton young bud and relate to fertility cotton due to transposition.