Characterization Of Transgenic Progenies With Antisense Suppression Of BnTT5 And BnTT8 Gene Families In Brassica Napus

Since the yellow-seeded Brassica napus line was screened out, it has been paid much and extensive attention to. In rapeseed, the yellow seeds have many good properties compared to the black seeds, such as thinner seed coat, less seed coat pigment, lower meal fiber content, higher seed oil content and meal protein content, etc. Presently, the germplasm of yellow-seeded B. napus is narrow and there are few yellow-seeded varieties. It is one of the important objectives to breed yellow-seeded rapeseed lines with stably and simply inherited yellow seed trait.Flavonoids exist in plants ubiquitously. They are the important plant secondary metabolizing products and are the main pigment components in Cruciferous plants. In Arabidopsis thaliana, the weakening of the flavonoids metabolizing may result in the seed color changing from dark brown to yellow or grey-brown. Such mutants in seed color are defined as transparent testa (tt). Now,22 TT loci have been identified and many of the TT genes have been cloned in A. thaliana.Chalcone isoenzyme (CHI, TT5) is the second pivotal enzyme in flavonoid pigment biosynthesis pathway in A. thaliana. It plays an important role in the synthesis and accumulation of anthocyanins and proanthocyanidins. In A. thaliana, the TT8 gene encodes a positive regulative factor belonging to the bHLH transcription factors. It acts with TT2 and TTG1 to form a tri-complex and regulates the expression of the terminal structure genes DFR and BAN etc. Consequently, it dominates the accumulation of anthocyanins and proanthocyanidins.In the present study, the transgenic B. napus progenies with antisense suppression of BnTT5 and BnTT8 gene families were widely characterized. The results are as follows: (1) The DNA Southern blot results of the transgenic accessions of the antisense BnTT5 suppression showed that the antisense BnTT5 T-DNA segment had been integrated into the B. napus acceptor genome successfully. Transgenic progeny seeds showed a trend of lightening when compated with non-transgenic CK, indicating that BnTT5 genes practically participate in determining the biosynthesis of seed coat pigments in Brasica species like rapeseed. The flowers of all BnTT5-antisense transgenic progenies showed little change, with still normal golden color. So whether the target BnTT5 genes antisense-suppressed in this study have any relations to rapeseed flower color formation deserves further study. Most other morphological traits and growth have no distict difference from the CK.(2) The DNA southern blot results of the transgenic accessions of the antisense BnTT8 suppression indicated that the antisense BnTT8 T-DNA segment had been integrated into the B. napus acceptor genome successfully. Semi-quantitative RT-PCR detection showed that the internal BnTT8 transcription had tissue-specificity. It showed some degree of suppression in the transgenic seeds of 35 d after flowering as compared with the CK, but the inhibition degree is weak and varied. The seeds of GUS-staining positive plants showed obvious lightening when compared with the CK. Though the transgenic seeds were not standar golden yellow, they were more yellowish than as black types. Most morphological traits of BnTT8-antisense transgenic progenies and growth have no distict difference from the CK, but the transgenic seeds generally showed decrease in seed-filling degree, with seeds deviated from standard ball-shape and decrease of 1000-seed weight from 3.15 g to 2.54 g on average. This study indicated that the BnTT8 genes might also participate in determination of some other seed traits besides seed pigmentation.(3) Results of GUS-staining of leaves of transgenic T1 and T2 plants, of embryos of pollen culture derived from T1 plants, and cotyledons and hypocotyls of T3 plants all showed that the transgenic plants conformed to Mendelian inheritance. Transgenic progenvies could successively survive and the transgenes together with the transgenic traits modifications could also be stably inherited.(4) In Southern detection, each independent transgenic line with antisense suppression of BnTT5 showed 1-2 hybridization bands. In most cases, the pollen plants from corresponding lines showed segregation ratios of GUS staining positive over negative reactions were near to 3:1. The T3 plants showed a ratio near 15:1 of GUS positive over negative reaction. The results conform to the segregion retios of 2 independent alleles, implying that possibly there were about 2 transgenes integraged into the genome of most antisense BnTT5-transformed lines, which were more that the Southern band numbers. In Southern detection, each independent transgenic line with antisense suppression of BnTT8 showed 0-1 hybridization band, and the GUS staining of the progenies conform to the segregation ratios of one independent alle. Hence, antisense BnTT8-transformed lines generally showed single-copy integration of the foreign construct, but antisense BnTT85-transformed lines generally showed double-copy integration of the foreign construct. The reason needs further study.(5) Antisense transformations of BnTT5 and BnTT8 both showed inhibition of the target genes, but they showed difference in suppression degrees. BnTT5 gene family has much more numbers (about 8) than BnTT8 gene family (about 2), and sequence divergence is also much higher. According to the results of this study, for polyploidy species like B. napus, gene silencing methodologies like antisense RNA are more effective on suppression of mini gene family containing members with high sequence similarities and less effective on gene family containing members with distinct sequence divergence. Furthermore, in this study, all transgenic progenies showed considerable remaining of target gene expression and target traits, and variations among plants, seeds and environments were also considerable. So in the future in transgenic molecular breeding for polyploid species like rapeseed, stronger gene silencing tools are needed to reach more optimal effect of suppressing internal gene expression and trait modification, and cautions should also be paid on the variations of transgene expression and environmental influence.