| In the preliminary study of this subject,the genes derived from the embryonic late-stage embryonic protein-like protein(Gene ID: 112292083,named the gene DEHY)from Physcomitrella patens were transferred to Ganyu 1(H1).A large number of positive transgenic lines were obtained from three varieties of upland rice,such as Ganyu 2(H2)and Drought 3(HN3).This project is to screen and further analyze the homozygous lines of transgenic rice transgenic offspring.The main results are as follows:1.In the field and laboratory,the transgenic lines of different days were screened by spraying a certain concentration of herbicides,and a certain number of individual plants were selected for the whole transgenic lines after spraying the herbicides,and the target gene PCR was carried out.A batch of candidate homozygous lines were identified by screening.The specific screening results were as follows: DEHY transformed H1 to obtain 8 and DEHY to transform H2 to obtain 20,and DEHY transformed HN3 to obtain 17 candidate homozygous lines.2.Southern blotting was used to perform copy number analysis on the candidate homozygous lines,and two single-copy lines(numbered 5-37,5-46),six two-copy lines,and 12 multiple copies were screened.Strains.The expression levels of the target genes were analyzed by semi-quantitative RT-PCR for single-copy and two-copy strains.The results showed that the expression of the target genes of the two single-copy lines was significantly higher than that of the two-copy lines.4.Using a certain concentration of PEG and NaCl stress,the stress resistance analysis in the seedling stage was carried out in the laboratory.The results showed that under the condition of NaCl stress,the survival rate of 5-37 strains was the highest after rehydration,reaching 36%.Under PEG stress The survival rate of 5-37 and 5-46 strains was the highest after rehydration,about 21%.Subsequently,the initial salt tolerance of transgenic lines was screened by 0.5% NaCl salt pool to obtain a highly salt-tolerant transgenic strain.The line is 5-37 strains.5.Perform TAIL-PCR insertion site analysis on the above 1-2 copies of the transgenic lines,and confirm that the DEHY gene has been stably integrated into chromosome 6 of the transgenic line 3-18 genome.Several genes in the 10 Kbp downstream of the target gene insertion site were analyzed,and the wild-type and 3-18transgenic lines were used for quantitative PCR analysis of these genes.The results showed that the gene downstream of the insertion site was Os06g0663900.The expression level was significantly lower than that of wild type plants. |
