The Study On The Relationship Between Lignin Biosynthesis Manipulation And Brassica Napus' Resistance To Sclerotinia Sclerotiorum And Lodging

Sclerotinia sclerotiorum and lodging are two important factors affecting oilseed rape production in China. They not only decrease oilseed yield, but also reduce the qualities of the processed oil and harvest index, and in practice things are worsened by a synergic mechanism between them which might put oilseed rape production at a greater peril. Some traditional technologies such as cultivation measurements and chemicals can be used to combat against the negative effects brought on by them to some degree, but by an ineffective or contaminative way. Though traditional breeding strategies and transformation of OXO, chitinase and β -1, 3-glucanase genes could advance S sclerotiorum resistance for Brassica napus partly, the S sclerotiorum resistant breeding has still not made substantial progress because of absence of resistant resources. For the study of lodging, it is just beginning in and out of China. So it is of a great significance to explore a new road for the improvement of disease and lodging resistance of oilseed rape and other crops.As an important component of plants' mechanical tissues and cell walls, lignin plays a great role in fortifying plants against many adverse environments such as wind, rain and pathogens. Being at a central position in lignin biosynthesis, 4-CL can conduct metabolic intermediates into lignin biosynthesis pathway, and is also taken as an ideal target gene for manipulating lignin biosynthesis. In this study, we first analyzed the correlation of lignin content and S sclerotiorum and lodging resistance of different B. napus cultivars and this would lay groundwork for the improvement of disease and lodging resistance through the increase of lignin content by the way of engineering technology. A 4-CL cDNA was isolated from Populus tremuloides by the method of homologous sequence and the biochemical function of the gene was analyzed. Furthermore, through the over-expression of the heterogeneous 4-CL gene in B. napus, we studied the effects of altered 4-CL activity on lignin content, S sclerotiorum and lodging resistance for the transgenic crop. The work was hoped to lay a foundation for yield, quality and adversity resistance breeding of B. napus and to provide a new viewpoint and way for the study and breeding of disease and lodging resistance of other crops. In another part of the study, we made an intercross with a specific S sclerotiorum and lodging resistant cultivar and a sensitive one and localized the QTLs associated with the resistant traits based co-dominant maker SSR and gene extrons oriented marker SRAP. And it was hoped to be helpful for the isolation and clustering of the genes in future. The results obtained were primarily as follows.1. The correlation analysis between lignin content and S sclerotiorum and push resistance was conducted on 29 B. napus cultivars. The results indicated that lignin content was positively correlated with both S. sclerotiorum and push resistance significantly, with correlation coefficients of 0.6971 and 0.618, respectively.2. A 4-CL cDNA was isolated from lignified stem tissues of Populus tomentosa. The DNA sequence analysis indicated that the cDNA contained transcription starting and ending codons and a full open reading frame (ORF). The deduced protein sequence contained two characteristic sequences of all known 4-CL proteins, namely box Ⅰ (SSGTTGLPKGV) and box Ⅱ (GEICIRG) . Phylogenicanalysis showed that the Populns tomentosa 4-CL was classified into class I of the 4-CL proteins, and phylogenically more related to Populus tremuloides 4-CL1 which is involved in lignin biosynthesis.3. A protokaryotic expression vector pETL was constructed and a 4-CL recombinant protein with a Hisx6 tag at amino terminal was expressed in E. coli. Enzyme analysis showed that the recombinant protein possessed catalytic activity and could activate the three lignin biosynthesis associated substrates PA, CA and FA, with a preferential order PA>FA>CA. the recombinant protein showed no activity to SA.4. 4-CL recombinant protein with a Hisx6 tag was purified by the Ni2+ affinity method and 4-CL antibody was prepared with the purified recombinant protein as antigen. 4-CL antibody would be further used to analyze the expression of the heterogeneous gene in transgenic lines.5. Two plant expression vectors were constructed and transformed into Brassica napus cultivars, Zhongshaung No. 9 and 37301. A total of 56 plants with Kan resistance were obtained. Among them, 51 plants including 12 transformed with the pBI121:4CZ construct and 39 transformed with the pBI-C4H:4CL one were regenerated from 37301;5 plants including 2 transformed with the pBU2l:4CL construct and 3 transformed with the pBl-C4H:4CL one were regenerated from Zhongshuang No. 9.6. The PCR analysis showed that the ratio of positive transgenic plants was about 60%. Southern blotting analysis of the putative transgenic plants indicated that the target gene was integrated into the B. napus genome.7. Western blotting and resistance tests were conducted. The result showed that the heterogeneous 4-CL gene was expressed in some of the transgenic lines, and in these lines the lignin content increased and S. sclerotiorum and lodging resistance were improved, while in some of the transgenic lines the target gene was also silenced.8. A F2:3 population was produced from the intercross between Zhongshaung No. 9 and 37301. A Brassica napus linkage map containing 22 groups was constructed with SSR and SRAP markers. The total length of the map was 1235.3 cM, and the average map distance between markers was 8.76cM.9. QTL analysis was conducted for S. sclerotiorum resistance. Three QTLs which accounted for 34.54% of total variance were identified to be associated with the resistance trait in JingZhou, and they were located on the linkage group 4, 15, 21 respectively. In XinZhou, four QTLs were detected to contribute to the resistance phenotype and were located on the linkage group 3, 13, 15, 21 respectively. They could explain the 36.19% of variance. The common QTLs detected in two regions were rl5 and r21 which in all accounted for 27.29%, 23.48% of variance in JingZhou and XinZhou respectively.