Rice Agriculture And Forestry On The 8th, The Agriculture And Forestry On The 8th M Cdna Library Construction And Bsl Gene Screening

A chemical lethal rice mutant Norin 8 m earring the bentazone sensitive lethal gene was induced from Norin 8 by 60Co gamma-ray radiation. The results indicated that bentazone sensitive lethal gene was controlled by a single recessive gene . The Norin 8 m seedling can be killed by bentazone (above 0.05%), but its FI seedlings are safe. Furthermore, the gene has no negative effects on agronomic traits no matter whether it is homozygous or heterozygous. The techniques for transferring and selecting the marker gene in breeding program and its application ensure the purity of hybrid seed. A making system of the two-line hybrid rice had been put forward.About 50,000 Norin 8m seeds were divided into 12 parts. The Norin 8m were treated with 0, 200, 225, 250, 275, 300 Gy CO60 and 0, 2000, 2300, 2600, 2900, 3200 X 2. 6X 1013N+/cm2 law energy ion bean implantation respectively. At the 2-leaf stage, all plants were cultived with 0.05% bentazon, the function of mutant gene can't be recovered.Total RNAs were extracted from seedling of Norin8 and its 60CO gamma-ray irradiation mutant Norin8 m during 2-3 leaves. The messenger RNA was isolated from the total RNA, the Norin 8 and Norin 8 m rice cDNA libraries were constructed. The primary libraies had a total of 1.2 106 phages for Norin 8 and a total 1.4 106 phages for Norin 8 m, 28 randomly selected clones were evaluated. Insert fragments by PCR amplification using T3, T7 primer were obtained and ranged from 400bp to 3kb with an average of 1kb. The 99% phages were recombinants by x-gal/IPTG. All of the above mentioned accorded with the general requirement of cDNA library construction .The specific primers were designed based on the sequence of the BSL marker obtained by RAPD . About 1kb lengths of digoxigenin-labeled DNA probes were used to detect BSL gene by dot hybridization. We screened Norin 8 rice about library 300,000 cDNA clones by plaque hybridization using probes. Some Positive clones were obtained. Meanwhile, the insert fragments were cloned and sequences were analyzed.