Cloning, Functional Analysis And Study For Epigenetic Regulation Of ZmCCT,a Major QTL Gene Conferring Resistance To Gibberella Stalk Rot In Maize

Stalk rot in maize is one of the most devastating soil-born diseases worldwide. In China, stalk rot in maize has recently become an epidemic disease in spreading trends, and the harm of stalk rot is increasing year by year. So, creation of maize cultivars with stalk rot resistance has become an important task. Dissecting the genetic architecture of stalk rot resistance and coloning the functional genes against stalk rot would provide theoretical basis for understanding of resistant mechanism in maize and molecular breeding. In our previous study, a major QTL-qRfg1 conferring resistance to Gibberella stalk rot has been delimited to a 170kb interval on bin 10.04 flanked by marker SNP551 and marker CCT11, ZmCCT then was selected as the candidate of QTL-qRfg1 by alignment of BAC sequnences and functional annotation. The main results of present study are listed as follows:1. Two TE-related PAVs (TE1 and TE2) are located in the QTL-mapping region of qRfgl.TE1-related PAV is located ?2.4 kb, while TE2-related PAV is located ?91 kb, from the ZmCCT start codon. A set of 47 maize material were evaluated for resistance to Gibberella stalk rot and genotype of TE1-related and TE2-related PAV. Result of association study indicated TE1-related PAV was more revelant to resistance of Gibberella stalk rot.2. ZmCCT from resistant inbred line 1145 was introduced into the susceptible maize hybrid Hill, four independent complementary transgenic events in T2, T4 and T6 generation were developed for resistant testing, the transgenic plants showed significantly enhanced stalk rot resistance of 8.2-37.5% in the field, as compared with their non-transgenic sibling plants. What's more, two independent RNAi transgenic lines in BC2F2 genertation were also developed for resistant test, transgenic plants showed significantly reduced resistance, with a decrease of 17.4-18.5%, relative to non-transgenic sibling plants. Both of complementary and RNAi transgenic tests demonstrated ZmCCT, regulating resistance to Gibberella stalk rot in maize, is the functional gene of QTL-qRfg1.3. Two NILs, Y331-?TE with Non-TE1 Y331-ZmCCT allele and Y331-qRfg1 with intact 1145-ZmCCT alelle, were developed from advanced backcross recombinants. ZmCCT in Y331-ATE and Y331-qRfg1 showed rapid and transient response to pathogen infection, but not for TE1 ZmCCT allele in Y331. ZmCCT expression in Y331-ATE and Y331-qRfg1 increased briefly in response to F. graminearum challenge, peaking at 3 hpi and then reverting to a basal level by 6 hpi, but expression of ZmCCT in Y331 slightly elevated.This result indicated the expression pattern of ZmCCT post pathogen inoculation is affected by TE1-realted PAV directly.4. A pair of NILs, Y331-ATE and Y331, were used to detect the DNA methylation staus within promoter of ZmCCT and its dynamic response to pathogen challenge. The promoter of Non-TE1 ZmCCT allele in Y331-ATE was hypomethylated, as compared with TE1 ZmCCT allele.DNA methylation frequency within promoter of Non-TE1 ZmCCT increased rapidly at 3 h after pathogen infection, but not for TE1 ZmCCT alelle. This result indicated the DNA methylation staus within promoter of ZmCCT and its dynamic response to pathogen challenge were up to TE1-related PAV.5. Y331-ATE and Y331 were also used to check the histone methylation within promoter of ZmCCT and its dynamic response to pathogen challenge. The promoter of Non-TE1 ZmCCT in Y331-ATE showed a bivalant chromotin, but only repressive histone marks were present in TE1 ZmCCT of Y331. After F. graminearum inoculation, active mark H3K4me3 in Non-TE1 ZmCCT decreased gradually, while the repressive marks H3K27me3 and H3K9me3 decreased briefly, then reverted rapidly. For the TE1 ZmCCT alelle in Y331, both active and repressive histone marks showed little change.6. The function of ZmCCT for flowering time and related traits was confirmed by checking complementary transgenic plans. Y331-ATE and Y331 were used to detect the photoperiod-sensitive expression pattern and status of DNA methylation within promoter of ZmCCT, the results indicated DNA methylation staus within promoter of ZmCCT in leaves,affecting flowering time and related triats in maize maybe via influcing the photoperiod-sensitive expression pattern of ZmCCT, were decided by TE1-related PAV.7. The pair of NILs, Y331-ATE and Y331 were used to identify the pleiotropic roles in tolerance to low-nitrogen and salt stresses. Y331-ATE with Non-TE1 ZmCCT showed better growth potential under abiotic stresses, as compared with Y331.8. Complementary transgenic plans with sibling controls were used to look for the inner connection between the photoperiod sensitivity and stalk rot resistance of ZmCCT. ZmCCT in leaves showed strong photoperiod sensitivity, but the transcription of ZmCCT in roots was only induced by pathogen infection resistance in roots and not by photoperiod. Transgenic plans showed stable resistance to Gibberella stalk rot under different photoperiods.