Cloning And Functional Identification Of Chalcone Isomerase Gene Families From Brassica Species

The yellow rapeseed seed shows many quality advantages, such as lower seed pigments and fiber content, and thinner seed coat, higher protein and oil content than black seed varieties in the same genetic background. Moreover, the seed coat color was impacted by environment. Breeding of yellow seed material by hybridization needs long cycle and is low in efficiency.Chalcone isomerase (CHI) is the second key enzyme of flavonoid pathway, participating in catalyzing the four hydroxyl chalcone become naringenin, used for synthetic anthocyanidins glucoside, procyanidins, flavonols, isoflavone, orange ketone, yellow ketone, polyurethanes and so on. They were important physiological role in during the growth, such as cross pollination, disease resistance, stress tolerance, yeild and quality. In A. thaliana, the CHI gene is identified as tt5 locus. Because of chalcone isomerase gene was invovled in loss of activity of wild-type seed coat color from dark brown to yellow.Moreover, there are "triple" relationships between the genomes of A. thaliana and Brassica according to the relationship of Yu’s triangle of Brassica species. It is possible to research genome evolution, crop origins and character formation, and will provide the new evidence to the important functions of Brassica gene cloning and functional sites with comparative genomic studies. In this research, used for technology of RACE, full-length cDNAs of the B.napus and B. rapa and B. oleracea CHI gene family were isolated, and genomic sequences were also obtained, respectively, The results of the Southern hybridization shoed also further evidence of CHI genes in these species copy number. RT-PCR detection showed that there is similarity between the CHI gene family in Brassica and A. thaliana CHI organ-specific genes. However, the differentiation between the members is very large. Southern blot showed that they were single or double copy integration. The plant expression vector RNAi was constructed by the framework of PFGC5941M, and transformation the black-seed line of Zhongshuang 10 and obtaining of the positive antisense-BnCHI transgenic plantlets.1) Systemic cloning of full-length cDNAs and genomic sequences of CHI gene families from B. napus, B. rapa and B. oleracea.Using the mixed total RNA from various reproductive organs and total genomic DNA as materials, full-length cDNAs and genomic sequences of 14 member genes of CHI gene families were isolated from B. napus, B. rapa and B. oleracea based on Rapid Amplification of cDNA Ends (RACE) and PCR technologies.The B. rapus CHI(BnCHI) gene family. BrCHI1, BrCHI2, BrCHI3 and BrCHI4 genes are 2093, 1954,1761 and 1483 bp, with typical-type mRNAs (not including the poly(A) tail, and the same for the following) of 1140,1120,1011 and 983 bp, respectively.The B. oleracea CHI (BoCHI) gene family. BoCHI1, BoCHI2, BoCHI3 and BoCHI4 genes are 1945,1941,1761 and 1516 bp, with typical-type mRNAs of 1034,1119,1016 and 986 bp, respectively.The B. napus CHI (BrCHI) gene family. BnCHI1, BnCHI2, BnCHI3, BnCHI4, BnCH16 and BnCHI7 genes are 1856,1937,1895,1762,1761 and 1483 bp, with typical-type mRNAs of 1042,1113, 1042,1012,1016 and 983 bp, respectively.2) Features of proteins encoded by Brassica CHI gene familiesAccording to the size of 14 Brassica CHI proteins, they can be divided into two groups. One group is 234～252 aa, the other is 304 aa, with molecular weight of 25.56～27.24 kDa and 32.92～33.36 kDa, respectively. The isoelectric points of BrCHI3/BnCHI4 and BoCHI3/BnCHI6 both are 7.68. the other being 4.84-6.96. AtCHI is aacidic protein, and most of Brassica CHI proteins are acidic, but some are basic.The evolutionary relationship of Brassica CHI proteins and AtCHI are larger differences with nucleotide in Brassica. CHI proteins are divided into two groups in Brassica, the similarity and consistency of members in the same groupis are higher than between the two groups. The consistency of 14 CHI proteins in Brassica is 50～100% and the similarity is 55.8～100%; relative to Arabidopsis AtCHI protein, consistency is 54.1-80.3%, similarityis 59.9～85.1%.According to SOPMA,αhelix is largest proportion (35.53-44.05%), followed by random coil (28.57-33.88%) and extended strand (17.53-21.71%) andβturn (5.58%-10.32%) in BrCHI, BoCHI, BnCHI proteins.According to Swiss-Model prediction, BnCHI, BrCHI and BoCHI proteins look like a bouquet upside down, a large and negative by theβ-sheet and three shortβ-sheet chains connected together to form a layer ofα-helix core structure.3) Evolutionary implications of Brassica CHI gene familiesThe corresponding relations of CHI genes-between B. rapa and B. napus:BrCHI1/BnCHI3, BrCHI3/BnCHI4, BrCHI4/BnCHI7. The corresponding relations of CHI genes between B. oleracea and B. napus:BoCHI1/BnCHI1, BoCH12/BnCHI2, BoCHI3/BnCHI6. Protein phylogenetic analysis further confirmed that BnCHI3, BnCHI4, BnCHI7 are derived from Chinese cabbage BrCHI1, BrCHI3, BrCHI4 and BnCHI1, BnCHI2, BnCHI6 are derived from the cabbage BoCHI1, BoCHI2, BoCHI3.Theoretical expectations, there should be correspondence between BrCHI2/BnCHI5 and BoCHI4/BnCHI8. However, this study we did not cloned the complete sequences of BnCHI5 and BnCHI8. But the similarities among BrCHI5, BoCHI2 and BnCHI2 are up to 96.51%; the similarities among BoCHI4, BrCHI4 and BnCHI7 are up to 99.00%. BnCHI5 and BnCHI8 in B. napus may be lost for some reason, or perhaps they have not been really deleted but just not cloned in this study.This study shows that there are 4,4.6-8 CHI genes in B. rapa, B. olerace and B. nupas resepectively. Numbers of genes, multiple alignments. and phylogenetic analysis all showed that CHI was triplicated in the ancestor of Brassica. and then a member was further duplicated. So, Clues from CHI loci fully support that B. napus is a tetraploid species of B. rapa and B. oleracea, and supports the Brassica "Triplication" assumption.4) Brasica CHI genes are mainly expressed in reproductive organs, while differences exist among species and gene membersThe total expression of BrCHI gene family is detected in all 12 organs, but there are some differences among organs. BrCHI1 is mainly expressed in reproductive organs. BrCHI2 is mainly expressed in developing seeds; BrCHI3 expresses in flower, bud and developing seeds; BrCHI4 is similar to BrCHI1, but significantly higher in seed development.BoCHI gene family is similar to BrCHI family. BrCHI1 is mainly expressed in reproductive organs; BrCHI2 is mainly expressed in developing seeds; BrCHI3 is expressed in flower, bud and developing seeds; BrCHI4 is similar to BrCHI1, but the expression level is lower.BnCHI genes are expressed in various organs like BrCHI and BoCHI. BnCHI1 and BnCHI3 are mainly expressed in reproductive organs. BnCHI2 is mainly only expressed in flowers and seeds. BnCHI4 and BnCHI6 are expressed in flower, bud and developing seeds, and the expression in young seed is very low. BnCHI7 is similar to BnCHIl, but the expression level is lower.5) Construction of RNAi vector of Brassica CHI gene familyThis study cloned the a 761-bp RNAi fragment BCHII from B. napus targeting Brassica CHI gene family, and constructed the 11674-bp RNAi vector pFGC5941M-BCHII (simplified as pBCHII) using a modified pFGC5941 as the backbone. The recombinant vector was verified by molecular identification and transformed into Agrobacterium tumefaciens strain LBA4404 to form engineering strain.6) Brassica CHI gene family shows characteristics of rapid evolutionMulti-alignment of Brassica CHI genes and deduced CHI proteins showed that there is a Plant Chalcone Isomerase Domain in BrCHI, BoCHI, BnCHI proteins. Brassica CHI genes obviously can be divided into two types and four sub-types. Homologies within groups are much higher than among groups, which are much higher than between Brassica CHI proteins and AtCHI. Multiple alignments of genes and phylogenetic analysis of proteins show that CHI genes in Brassica are originated from an ancestor Cruciferous CHI gene.On the basic properties of the protein analyses, BrCHI3, BnCHI4, BoCHI3, and BnCHI6 are basic proteins, with pIs of 7.68; the rest Brassica CHI proteins are acidic with pIs of 4.84～6.96.RT-PCR results suggest that BrCHI1 may be mainly involved in the synthesis of flower color and seed coat pegment; BrCHI2 is mainly express in early developing seed and may be mainly involved in the formation of seed coat pigment; The function of BrCHI3 maybe diverge from others and play an role in response to stress; BrCHI4 is similar to BrCHI1, but the expression in developing seed is significantly higher.BoCHI1 may be mainly involved in the synthesis of flavonoids affecting the color of flower and seed coat; BoCHI2 may be affected by UV-induction or other factors, mainly involved in the formation of seed coat color; BoCHI3 is expressed in the leaves, flowers, buds and seeds, and expression in immature seeds is very low. its function may be involved in plant stress tolerance; BoCHI4 is similar to BoCHI1, but its expression in the mid-stage developing seed is higher. BnCHIl and BnCHI3 may be involved in seed coat pigmentation. BnCHI2 is expressed in seed, and might be affact by ultraviolet or other factors for plant self-protection. BnCHI4 and BnCHI6 expressions are higher in immature seed than in buds, flowers and late-stage developing seed. Furthermore, the expression of BnCHI4 is higher than BnCHI6.7) RNAi transformation results indicate that CHI genes simultaneously influence seed filling and seed pigmentation in BrassicaThe 751-bp RNAi fragment of Brassica CHI gene family was cloned. Uing pFGC5941M as platform vector, the RNAi vecotor was constructed and named pBCHII. Using improved leaf-disc method, it was used to transform the typical black-seeded cultivar Zhongshuang 10.10 regenarated plants were obtained. After Basta screening and PCR check,9 transgenic plants were identified. RT-PCR detection of 20d seeds revealed that the expression of all BnCHI member genes were down-regulated in transgenic plants to certain extent. Extent of down-regulation of each member correlates to its homology to the RNAi fragment. Compared with black-seeded CK, transgenic seeds were yellow-brown with distinct reduction of seed coat pigmentation and also affected in seed filling degree. Transgenic plants have limited or little change in other traits.This is the first time of systemic cloning, functional dissection and comparative study of CHI family genes in Brassica, which also shows potentials in mechanism study and molecular breeding of flavonoid traits in Brassica.