| Chinese kale(Brassica oleracea var. alboglabra Bailey) belongs to the Brassicaceae family. Chinese kale is widely distributed in southern China, Taiwan, Japan and Southeast Asia. Chinese kale is a rich source of antioxidants and anticarcinogenic compounds, including vitamin C, glucosinolates, carotenoids and phenolic compound. Glucosinolates(β-thioglucoside-N-hydroxysulfates) are nitrogen- and sulfur-rich plant amino acid-derived secondary metabolites. Glucosinolates and their hydrolysis products have important biological activities such as crop protectants, flavor precursors and cancer-prevention agents. Glucosinolates encompass three major chemical classes, aliphatic, aromatic and indole glucosinolates, based on their precursor amino acids and R group modifications. Glucoraphanin(4-methylsulfinylbutyl glucosinolate), an aliphatic glucosinolate, is the glucosinolate precursor of the bioactive isothiocyanate sulforaphane. Glucoraphanin is abundant plant secondary metabolite found in cruciferous plants,which shows strong evidences of anticarcinogentic, antioxidant and antimicrobial activities.This study was carried out with Chinese kale of different glucoraphanin content as material. Three structural genes involved in glucoraphanin metabolism, branched-chain aminotransferase 4(BCAT4), methylthioalkylmalate synthase 1(MAM1) and dihomomethionine N-hydroxylase(CYP79F1), were cloned from Chinese kale. Three regulated genes involved in glucoraphanin metabolism, MYB28, MYB29 and MYB76, were cloned from Chinese kale. Sequence homology and phylogenetic analysis identified these genes and confirmed the evolutionary status of Chinese kale. The expression patterns of the target genes were assayed by quantitative real-time PCR(q RT-PCR). Simultaneously, the extraction and analysis of glucosinolate provided a better understanding of the roles of structural genes(BCAT4, MAM1, CYP79F1) and regulated genes(MYB28, MYB29, and MYB76) in the glucoraphanin biosynthesis of Chinese kale. In this study, over-expression and RNAi plant expression vector of MYB28 were constructed. Then they were transformed into Arabidopsis thaliana with floral dipping method. At the same time, they were transferred into Chinese kale using Agrobacterium tumefaciens as intermediate. The content of aliphatic glucosinolates and transcript levels of aliphatic glucosinolate biosynthetic genes were detected in over-expression and RNAi lines. The researeh provided material basis for inereasing the glucoraphanin content in Chinese kale through genetic engineering. The main results are as follows:(1) The full-length c DNA of BCAT4(Genbank accession number: KP295464), MAM1(Genbank accession number: KP295465), CYP79F1(Genbank accession number: KP295466), MYB28(Genbank accession number: KP723785), MYB29(Genbank accession number: KP723786) and MYB76(Genbank accession number: KP723787) have been cloned from Chinese kale. Sequence analysis of BCAT4 indicated that the full length c DNA was 1394 bp including 42 bp 5?-untranslated region, 1143 bp open reading frame(ORF) and 209 bp 3’-untranslated region. The nucleotides encoded 380 amino acids with a predicted molecular mass of 42 k D and isoelectric point of 9.09. The full-length c DNA sequence of MAM1 was composed of 1560 bp. The c DNA sequence contained an 1164 bp open reading frame that encoded an estimated polypeptide of 387 amino acids with a molecular weight of 42 k Da and an isoelectric point of 5.99. The full length MAM1 c DNA also consisted of 291 bp 5’-untranslated region and 105 bp 3’-untranslated region. Analysis of the nucleotide sequence indicated that the c DNA of CYP79F1 was 1685 bp with an ORF of 1626 bp. The full-length c DNA comprised 27 bp 5’-untranslated region and 32 bp 3’-untranslated region. The deduced CYP79F1 protein was a polypeptide of 541 amino acid with a putative molecular mass of 61 k Da and a p I of 8.26. Analysis of the nucleotide sequence indicated that the c DNA of MYB28 was 1257 bp with an ORF of 1020 bp. The full-length c DNA comprised 138 bp 5’-untranslated region and 99 bp 3’-untranslated region. The deduced MYB28 protein was a polypeptide of 339 amino acid with a putative molecular mass of 38 k Da and a p I of 6.87. Analysis of the nucleotide sequence indicated that the c DNA of MYB29 was 1081 bp with an ORF of 966 bp. The full-length c DNA comprised 24 bp 5’-untranslated region and 91 bp 3’-untranslated region. The deduced MYB29 protein was a polypeptide of 321 amino acid with a putative molecular mass of 84 k Da and a p I of 5.07. Sequence analysis of MYB76 indicated that the full length c DNA was 1066 bp including 23 bp 5?-untranslated region, 1017 bp ORF and 26 bp 3’-untranslated region. The nucleotides encoded 338 amino acids with a predicted molecular mass of 88 k D and isoelectric point of 5.06.(2) Quantitative real-time PCR(q RT-PCR) were used to analysis the expression levels of glucoraphanin biosynthetic genes(BCAT4, MAM1, CYP79F1, MYB28, MYB29, and MYB76) in different cultivars, different organs and different developmental stages. The V results indicated that the glucoraphanin biosynthetic genes were expressed mainly in cotyledon, leaf and stem, while the transcript level of glucoraphanin biosynthetic genes were higher in cotyledon, leaf and stem compared with flower and silique. The glucoraphanin biosynthetic genes were expressed throughout leaf development with lower transcript levels during the younger stages. The glucoraphanin biosynthetic genes had relatively high transcript accumulation in the mature and inflorescence leaves compared with the primary and young leaves. The expression levels of glucoraphanin biosynthetic genes were consistent with the glucoraphanin accumulation in eight cultivars of Chinese kale. The expression levels of BCAT4, MAM1 and MYB28 were higher at vegetative–reproductive transition phase with low glucoraphanin content than those at reproductive phase with high glucoraphanin content. The expression levels of CYP79F1, MYB29 and MYB76 were lower at vegetative–reproductive transition phase than those at reproductive phase.(3) The specific primers were designed according to the c DNA sequence of MYB28 in Chinese kale. The interest fragment, about 1200 bp, was amplified by PCR, and then was linked to T-vector. The clone vector and expression vector p FGC5941 was digested by Bam HI and Sma I. The target fragment was linked to p FGC5941 to get the over-expression vector. Then it was transformed into Agrobacterium EHA105 for transformation. In this study, based on the c DNA sequence of MYB28 in Chinese kale, the specific primers were designed to amplify the specific fragment with 270 bp. The target fragment was linked to the p FGC5941 vector in opposite to get the RNAi vector. Then it was transferred into Agrobacterium EHA105 for transformation.(4) The overexpression and RNAi vectors were transformed to Arabidopsis thaliana by floral dip transformation method. The over-expression and RNAi vectors were transferred into Chinese kale using Agrobacterium tumefaciens as intermediate. PCR detection and Southern blot analysis showed that the target gene had been integrated into Arabidopsis thaliana and Chinese kale. The expression patterns and transcript levels of aliphatic glucosinolate biosynthetic genes were assayed by q RT-PCR. The contents of aliphatic glucosinolates were carried out by high performance liquid chromatography(HPLC). The results indicated that over-expression and RNAi lines of Arabidopsis thaliana and Chinese kale showed no visible difference on plant morphology. The transcript levels of aliphatic glucosinolate biosynthetic genes and the content of aliphatic glucosinolates were increased in over-expression lines and decreased in RNAi lines. The content of glucoraphanin increased 97% in over-expression lines of Chinese kale compared with wild type. |
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