Chalcone Isomerase (GhCHI) Involved In Flavonoid Biosynthesis And The Regulation Of Growth And Development Of Cotton (Gossypium Hirsutum L.)

Cotton is one of the most important economic crops, provides a large number of high-quality raw materials for textile industry. Flavonoids, one of the secondary metabolites which are widely distributed in higher plants, play important roles in the growth and development of plants, They are the primary compounds of plant pigments, and involved in pollen germination, auxin transport and environmentally induced stress. Chalcone isomerase（CHI, EC 5.5.1.6） is the key enzyme in flavonoids biosynthetic pathway, catalyses the intramolecular and stereospecific cyclization of naringenin chalcone into naringenin.To reveal how GhCHI effect flavonoid biosynthesis and the regulation of growth and development of cotton, this gene was cloned on the basis of analysing biologic informatics. Next, the full-length coding sequence of GhCHI-A/D was inserted into pET-28 a and transformed into Escherichia coli strain BL21, the recombinant protein was purified and analysis this proteins characterization. The GhCHI-A RNA interference（RNAi） silencing and overexpression vectors were constructed to transform J14, study the effects of suppression of GhCHI on flavonoid synthesis and cotton growth and development.Main results as follows: 1. There exist two Type-I CHI proteins in cotton.Using AtCHI gene as the inquiry probe, we searched homologous sequences in upland cotton genome（AADD）. Phylogenetic analysis showed that, there have 14 GhCHIs in cotton that fall into three subfamilies, two of them（Gh A₁3G0197 and Gh D₁3G0211） belong to a legume-specific clade known as ‘Type-I’ CHIs. The alignment results showed that: this two genes shared highly similarity with Type-I CHI gene sequence and had complete substrate binding site and conserved amino-acid residues as well as specific substrate amino-acid residues of Type-I CHI proteins. Moreover, they had high similiarity with Ga CHI and GrCHI in genetic structure and gene sequences, named GhCHI-A and GhCHI-D, respectively.To determine the tissue-specific expression patterns of the GhCHI gene, qRT-PCR was performed. Our results showed that GhCHI was broadly expressed in cotton, and it was especially abundant in ovule and leaf, while almost undetected in 20-dpa and 25-dpa fibers. Moreover, strong expression of GhCHI was detected in half expansion leaf. 2. Activity detection of GhCHI proteins.To prove whether GhCHI protein has catalytic activity, we cloned the GhCHI ORF into the expression vector pET-28 a, which was transformed into E.coli BL21（DE3）. After induced by IPTG, fusion protein was purified using Ni–NTA affinity column. Experiments show that GhCHI was capable of catalyzing the isomerization of chalcone into naringenin, but doesn’t have the capable of degrading naringenin. An in vitro enzyme assay showed that GhCHI enzyme had optimum activity at pH 8.0 and 37℃, we got the enzyme kinetics parameters of this proteins under the optimum condition, the Km of GhCHI-A was 133.75 nM and the Vmax was 0.0137 nM/min, the Km of GhCHI-D was 211.10 nM and the Vmax was 0.0226 nM/min. Therefore, we concluded that GhCHI is a functional Type-I CHI protein. 3. GhCHI-A effect the accumulation of flavonoid in the cotton.To research the role of GhCHI in flavonoid biosynthesis pathway, we cloned GhCHIA gene from G. hirsutum, and obtained transgenic plants by transferring GhCHI-A RNAi and overexpression vectors into cotton, respectively. qRT-PCR analysis of GhCHI expression, showed an extemely low level of expression in RNAi plants, and highly expression in overexpression plants. At the same time, the catalytic activity of the GhCHI protein was significantly lower in the RNAi plants, and slightly higher in the overexpression plants. The RNAi plants compared with wide-type, the anthocyanidin content in leaf and petals（0dpa,1dpa） was significantly increased and quercetin accumulated in root tip region, PAs content in seeds was aslo increased. While for the overexpression plants, the anthocyanidin content in leaf decreased apparently, quercetin accumulated in root tip region and the amount of PAs in seeds didn’t show obvious changes. All mentionded above reveals that down-regulation of GhCHI-A can promoted the accumulation of flavonoids（anthocyanidin and quercetin）, while overexpression of GhCHI-A can retarded the accumulation of anthocyanidin in leaf. 4. Regulation GhCHI-A gene influence the growth and development of cotton.To further understand the role of GhCHI in cotton development, compared the differences of cotton growth, seed and fiber phenotypes of the wide-type and transgenic plants. RNAi plants were seriously shortened, the height of plants reduced by 15.5%, seeds hundred-grain weight and average length of upper fiber, 20% and 5.5% reduced in comparison with the control, the lint increased by 10%. However, overexpression plants had no change on the plants heigh, but the seeds hundred-grain weight and lint decreased by 12% and 12%, respectively, seeds are yellow, the average length of upper fiber increased by 18%, fiber strenth has dramatic change. In conclusion, both down-regulation and overexpression GhCHI-A can influence the growth and development of cotton plants and seeds.