Preliminary Study On Flowering Mechanism Of Early And Late Maturing Of Oilseed Rape(Brassica Napus L.)

Oilseed rape is one of the major oil crops, and also an ideal winter crop. The planting pattern of rape-rice rotation has been formed in China, but the longer growth period of popularized rape(Brassica napus L.) affects the planting of the subsequent rice. High temperature, rainy season and other adverse climatic conditions also affect the yield and quality of late-maturing rape. It is an important primary breeding objectives to breed high yield and good quality of early-maturing rape. Early or late mature period of rape is determined by flowering time. Flowering is one critical event in the transition from vegetative phase to reproductive phase and plays a very important role in plant life. It is very meaningful to study its mechanism. Although the mechanism of flowering has been studied for more than a hundred years, there is still not a systemic theory for various plants and it is necessary to study the mechanism of more plants, including rape.The major research contents of this dissertation were, (1)Physiological and biochemical changes during flower formation in rape with different maturing were studied with three cultivars RG-8M (early-maturing mutant of late-maturing rape RG-8),'Fuyou No.4'(medium-late maturing rape) and RG-8. (2)Flowering related gene LFY and CO of rape were cloned from RG-8M by rapid amplification of cDNA ends(RACE) technology. Their expression in different growth periods and different organs were preliminary tested. (3)Differential proteins in the leaf of vernalized and unvernalized rape were analyzed from RG-8M,'Fuyou No.4'and RG-8. The main results were as follows:1. The starch content in the leaf of three rape cultivars during flower formation appeared to be descending and the (α+β)-amylase activity(mainlyβ-amylase) and the soluable sugar content appeared to be ascending. This indicated that starch was hydrolyzed by amylase to soluable sugars, so that increased carbohydrate could promote the flower formation of rape. Moreover, the reducing sugar and fructose content in the leaf of rape during flower formation appeared to be ascending. This showed that reducing sugar and fructose with high level may be used as structural or energy substance to promote the flower formation of rape plants. The soluable sugar, reducing sugar and fructose content of RG-8M during flower formation was significantly lower than that of'Fuyou No.4'and RG-8. It showed that a relatively low sugar content favored early flowering of rape. The changes of sucrose content and the acid invertase activity of three rape cultivars during flower formation were different. It showed that sucrose may be used as energy and signal substance during the flower formation of rape. The high acid invertase activity of RG-8M during flower formation may be essential for its earlier flowering.2. The content of soluable protein and dissociate amino acid in the leaf of three rape cultivars was in a low level before floral bud differentiation and ascended differently after floral bud differentiation. It showed that the soluable protein and dissociate amino acid with low level was benefit for the flower bud initiation of rape plants, and the high soluable protein and dissociate amino acid content favoring flower formation provided material guarantees to meet nutritional requirement for the flower bud morphological differentiation and development of rape plants. The comparison result of three rape cultivars showed that the moderate high amount of soluable protein and dissociate amino acid was conducive for RG-8M to flower earlier. The nucleic acids (mainly RNA) and DNA content in the leaf of rape during the morphological flower bud differentiation increased at the same time. This indicated that there was a strong DNA replication and RNA synthesis process during this period, which could promote protein synthesis. It was benefit for flower bud differentiation of rape. Compared with'Fuyou No.4'and RG-8, RG-8M had more RNA content and a higher RNA/DNA of during flower bud differentiation. It showed that RG-8M had a more vigorous protein synthesis process guided by nucleic acid, and the synthesized specific proteins and enzymes promote the flower bud differentiation of RG-8M. The changes of RNase and DNase activity of three rape cultivars during flower formation were different, but they were kept low before budding period. It indicated that the catabolism of nucleic acids was weak, and the anabolism was dominant during this period. This result was beneficial for flower bud differentiation of rape.3. The GA3 content in the leaf of three rape cultivars decreased before floral bud initiation and increased after floral bud differentiation. It indicated that the low level GA3 was benefit for the flower bud initiation in rape, and the high GA3 content favored the flower bud morphological differentiation and development of rape. Three other hormones of rape during flower formation were studied. The results showed that low level IAA, high level ABA and ZR were benefit for floral bud differentiation. Each hormone content of RG-8M during flower formation was less than'Fuyou No.4'and RG-8, which showed that a relatively low hormone level was conducive for its earlier flowering. In addition, the high ABA/GA3 and ZR/GA3 ratio could promote floral bud differentiation of rape, especially flower bud initiation, while the high ABA/IAA ratio and low ZR/ABA ratio were in favor of flower formation. The IAAO activity negatively correlated with IAA content of rape during flower formation appeared to be descending and the trend of IAA content was the opposite. This indicated that the IAAO high activity was benefit for floral bud differentiation of rape and IAA of rape during flower formation was mainly degradated by IAAO. The POD activity low correlated with IAA content increased before floral bud initiation, which indicated that the low POD activity was in favor of the flower bud initiation of rape and the POD activity affected floral bud differentiation not through degradating IAA. The relative high POD activity of RG-8M may be essential for its earlier flowering.4. The PAs (mainly Put and Spd) content in the leaf of three rape cultivars decreased before floral bud initiation and increased after floral bud differentiation, but the rise and the time to peak of different species were quite different. RG-8M increased more quickly than'Fuyou No.4'and RG-8 and reached peak earlier. This showed that the PAs with low level was in favor of the flower bud initiation of rape and the increase of its content was benefit for the flower bud morphological differentiation and development of rape, and the PAs content of early-maturing rape reached the high level earlier. The PAO activity and the content of total polyamines in the leaf changed in parallel, and there was a significantly positive relationship between them. It showed that the high PAO activity was benefit for flower formation. The chlorogenic acid content in the leaf of three rape cultivars during flower formation appeared to be descending, which showed that the low level chlorogenic acid could promote the flower formation of rape, and the earlier-maturing rape required the higher relative content of chlorogenic acid for its flower bud differentiation.5. Degenerate primers based on conserved sequence of the cloned LFY homologous genes were used to isolate the LFY homologous gene fragment of rape from the genomes of RG-8M. Based on the homologous gene fragment, the full length cDNA of LFY homologue gene (named as BnLFY) was isolated with specific primers and RACE. The length of BnLFY cDNA sequence was 1310bp and included a 1248bp open reading frame (ORF) that encoded 415 amino acids. The deduced amino acid sequence of BnLFY gene shared with other dicotyledon LFY-like proteins special regions such as N terminal proline rich region, the central acidic domain, the leucine zipper structure and the lysine and arginine-rich basic region, which were some structural features of transcription factors. The BnLFY protein was over 85% identical to LFY-like proteins of several cruciferous plants. RT-PCR analysis showed the BnLFY gene was a constitutive gene in rape genome. The transcripts of BnLFY gene were detected in different growth periods and different organs.6. The full length cDNA of CO homologue gene (named as BnCO) was isolated with the method for cloning BnLFY. The length of BnCO cDNA sequence was 1348bp and included a 1281bp ORF that encoded 426 amino acids. The deduced amino acid sequence of BnCO gene shared with the complete structure of other CO-like proteins and contained highly conserved B-box zinc finger domain and the CCT domain, which indicated that it encoded a complete transcription factor. Because it contained two complete B-box motifs, the BnCO belonged to the first class of CO gene family. The amino acid sequence of BnCO gene shared highly homology with CO-like protein of Brassica napus, Brassica oleracea var. alboglabra, Brassica nigra and Sinapis alba, especially in the 3'end and 5'end. BnCO protein shared almost 100% identity with BnCoa1, Bn9CON10 and Bn1CON19 proteins of rape in these region, so these region might be essential for BnCO gene. BnCO gene shared highly homology with the cloned CO homologous genes of rape, but there was an additional sequence containing 60 amino acids in the middle of BnCO protein. Therefore, BnCO gene might be a new member different from other cloned members of CO gene family in rape. RT-PCR analysis showed the BnCO gene was a constitutive gene in rape genome. The transcripts of BnCO gene were detected in different growth periods and different organs.7. Proteomic approach was applied to compare the proteome changes in the leaf between three vernalized and unvernalized rape cultivars. There were 27 proteins differentially expressed, and 6 proteins were shared by three cultivars. Twenty-two proteins were successfully identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/MS) analysis and protein data searching. These proteins were classified into 6 categories according to their different function. They were involved in photosynthesis(45%), energy metabolism(14%), antioxidation(9%), protein and amino acid metabolism(14%), stress resistance(14%), and transport (5%). Rubisco(ribulose-1,5-bisphosphate carboxylase/oxygenase), Rubisco large subunit, putative PSⅠtypeⅢchlorophyll a/b-binding protein and triosephosphate isomerase were closely related with vernalization of rape, and they were also correlated with the maturity of rape. The maturity of rape was later, the difference in the expression of these proteins was more significant. The differential expression of carbonic anhydrase, 23 kD subunit, putative delta subunit of ATP synthase, AT2G37660, trypsin inhibitor propeptide, 60S acidic ribosomal protein P3, ribosomal protein L12-A and O-acetyl serine thiolase in RG-8M was completely different from that in'Fuyou No.4'and RG-8, which showed that these proteins might be associated with the early maturity of rape.