Quality Analysis And Studies On Relationship Of Genetic Resources Of Tai-Tsai

Glucosinolates, amino acid, the synthetic substrate of glucosinolates, and aroma components, the degradation products of glucosinolates, were studied Tai-tsai (Brassica campestris ssp. chinensis Makino var. tai-tsai Hort). CYP79B5, a key gene in the synthesis of glucosinolates was cloned and expressed in Tai-tsai. Moreover, classification of Tai-tsai resources was conducted using RAPD and ISSR makers. The results are as follows:1. Evaluation of Nutrition Component in Tai-tsaiComposition of amino acids in Tai-tsai were made clear by analyzing the components and contents of amino acids in 4 Tai-tsai varieties. The results showed that there were 17 amino acid (tryptophan can not be detected due to destruction), among which 7 amino acids were the essential amino acids. Moreover, the content of glutamate was the highest, while that of cysteine was the lowest. According to the standard proposed by WHO/FAO, the nutrition value of Tai-tsai can be improved by supplementing the content of methionine and cysteine. By comparison with the nutrition component of 8 varieties of Brassica, it can be concluded that the contents of amino acids in Tai-tsai were higher than Brassica chinensis and Brassica pekinensis, while the contents of soluble protein was between Brassica chinensis and Brassica pekinensis, and the contents of soluble sugar was lower than both Brassica chinensis and Brassica pekinensis, which can provide evidence for further study on quality study and germplasm utilization of Tai-tsai.2. Evaluation of Glucosinolate Composition and Contents in Tai-tsaiFour Tai-tsai cultivars, eg.'Jingyan Taicai','Nanjing Xiaoye Taicai','Huaye Taicai' and'Xiaoye Taicai'grown in different season i.e.spring and autumn were used to evaluate glucosinolate composition and contents in leaf blade and petiole of plants by HPLC analysis. The results showed that eight glucosinolate ingredients including Pro, NAP,4OH, GBN, NAS, GBC, NEO and 4ME were detected in all testing materials. It also showed that glucosinolate contents in leaf blade were higher than that in petiole. Moreover, glucosinolate contents in those grown in autumn were higher than that in spring. Great variation of glucosinolate contents were also detected among the four cultivars.3. Analysis of Volatile Components of Tai-tsai in Different CultivarsTo analyse the effects of cultivating season on the volatile components of Tai-ts ai and to provide further study on aroma compounds in Brassica vegetables with th eoretical basis, volatile components in three Tai-tsai varieties grown in spring and a utumn were analyzed by headspace-solid phase microextration and Gas Chromatogra phy-Mass spectrometry (SPME-GC/MS). Eleven categories of compounds include alc ohols, aldehydes, esters, ketones and nitriles were found, which varied in cultivars. And the compound quantity in those cultivated in autumn was more than that in sp ring. Moreover, (E)-2-butenedioic acid diethyl ester,1-Butene,4-isothiocyanato-,2,4-Hexadien-1-ol,3-Hexen-1-ol, (E,E)-2,4-hexadienal,2-Hexenal,3-Pentenenitrile, benzen-epropanenitrile and (2-isothiocyanato-ethyl)-benzene were those compounds with high contents. The volatile components in Tai-tsai were affected by cultivar and growing season.4. Molecular Cloning and Prokaryotic Expression of cyp79B5 from Tai-tsaiCYP79B5 gene was cloned from'Jinyantaicai'by RT-PCR. The full-length cDNA of CYP79B5 was 1847 bp long, with a 1620bp open reading frame (ORF) encoding 540 amino acids.2112 bp fragment was obtained by PCR amplification using genome DNA as template. Blasting results showed that the deduced amino acid sequence had high homology to Brassica napus, sinapis, and Arabidopsis thaliana. It had 100% identity with that of Brassica napus, which primarily proved that it was the full-length sequence of CYP79B5. Bioinformatics analysis showed that there was a significant protein signal domain in the deduced amino acid sequence, and a similar 3D structure could be found in the SWiSS-MODEL database. The complete coding sequence was amplified by PCR. Then the combinant plasmid pET-24a (+) was constructed, and transformed to Escherichia coli Rosetta (DE3) for expression induced by IPTG. SDS-PAGE analysis revealed that there was a specific band at 64.1 KD, which conformed to the expected molecular weight of the combinant protein. 5. Studies on germplasm resources of Tai-tsai using RAPD markersThe DNA polymorphism of twenty-nine Tai-tsai cultivars were analyzed using randomly amplified polymorphic DNA (RAPD) markers in the present study. RAPD reaction conditions was optimized. Thirty-three RAPD primers selected from 273 primers generated a total of 336 clear and reproducible bands, of which 293 were polymorphic, with an average of 10.2 polymorphic fragments per primer, The polymorphic rate was 87.2 percent, which showed that the genetic diversity of Tai-tsai was rich. A dendrogram was constructed by RAPD markers, and the distance coefficient was from 0.63 to 0.79. By the RAPD markers, the genetic relationship of Tai-tsai germplasm was made clear. These results would be useful for the protection and utilization of Tai-tsai germplasm resources in the future.6. Studies on germplasm resources of Tai-tsai using ISSR markersThe DNA polymorphism of twenty-nine Tai-tsai cultivars were analyzed using inter simple sequence repeat (ISSR) markers in the present study. ISSR reaction conditions were optimized, and the optimum condition was selected. Forty-six ISSR primers selected from 100 primers generated a total of 522 clear and reproducible bands, of which 414 were polymorphic, with an average of 9.0 polymorphic fragments per primer, The polymorphic rate was 79.3 percent, which showed that the genetic diversity of Tai-tsai was rich. A dendrogram was constructed by ISSR markers, and the distance coefficient was from 0.70 to 0.86. The indicated that there was little difference between varieties. By comparison of RAPD and ISSR markers, the genetic relationship of Tai-tsai germplasm was further discussed, these results would be useful for the protection and utilization of Tai-tsai germplasm resources in the future.