The Molecular Mechanism Of Short Catkin Mutant Related To Gibberellins In Castanea Mollissima Bl

Chinese chestnut(Castanea mollissima B1.) is a very valued plant species with an important role in food, economy and ecology. However, more male flowers and less female flowers significantly limits yield in Chinese chestnut. Thus, breeding and selecting chestnut trees with relatively more female flowers is a desirable work in improving chestnut yield. Fortunately, a novel natural bud mutant of Chinese chestnut was found, which was associated with a greater number of female flowers and increased yield. During the development of mutant short catkins, the upper parts became yellow, curved, and finally dropped off gradually, while the basal catkins still developed normally. The average length of mutant catkins was2.2cm. and this mutant catkin(MT) causes6to8-fold shorter catkins comparison of wild-type catkins(WT).This Castanea mollissima mutant has been registered in State Forestry Administration, P. R. China (Varieties No.20090015) and was named ’duanhuayunfeng’.Prophase research showed that short male catkin mutant is closely related to gibberellins, and the expression of ent-kaurenoic acid oxidase (KAO) in mutant catkins was significantly lower than that in WT during different developmental stages, and gibberellin content was lower too. In this study. cDNA sequence alignment of the gibberellin biosynthesis key enzymes ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO). KAO. GA20-oxidase1(GA20ox1) and GA3-oxidase1(GA3ox1) between wild type and mutant catkins, and the short male catkins was infected with the plant over-expression vector of KAO and KO, in order to find the molecular mechanisms that gibberellin content decreased in mutant catkins, and else provide support for the identification of the gibberellin-deficient mutant of the short male inflorescence, at the same time provide a theoretical basis for the regulatory mechanism of the chestnut production.The results were summarized as follows:1. Using quantitative real time PCR (RT-PCR), we further compared the expressions of representative GA biosynthesis-related genes in the primordium formation of the floral cluster, such as CPS, KS, KO, GA20oxl and GA3ox1in wild type and mutant catkins. Interestingly, the expression of KAO was down regulated in MT. In MT, the transcript level of KAO, encoding a protein that catalyzes the oxidation of ent-kaurenoic acid, was about0.5fold of the wild-type by an unknown mechanism. Whereas, the comparative expressions in WT and MT were analyzed and no significant difference of CPS, KS, KO, GA20oxl and GA3oxl were detected in the primordium formation of the floral cluster between the WT and MT. The lengh of DNA of KAO in Castanea mollissima B1. was6086bp, there were8exons,7introns. The promoter of KAO was cloned by Tail-PCR, and the T of WT convert to A of MT at-251upstream translation start sites.2. The cloned nucleotide sequence of KAO cDNA inserted into restriction site BglⅡ and PmlⅠ of plasmid pCAMBIA1304, constructing the plant over-expression vector pCAMBIA1304-CmKAO. Using agrobacterium-mediated transformation system, the vector carrying KAO gene were evenly injected throughout the short catkins by using a sterile1mL hypodermic syringe and the short catkins were soaked with writing brush soon in the primordium formation of the male inflorescence. A transient expression system of the chestnut catkin was established.3. Catkins of mutant catkin (MT) and mutant catkins that transformed with the pCAMBIA1304empty vector (MT+EV) began to appear on PCD20days after infection, and mutant catkins which transformed with the pCAMBIA1304-KAO (MT+KAO) did not occur, and the lengh of MT+KAO was2-3times of MT, MT+EV. CaMV35S was-verified in MT+EV and MT+KAO; it showed that catkins had been infected by Agrobacterium. Real-time PCR and semi-quantitative RT-PCR analyses were carried out with MT, MT+EV and MT+KAO. The expressions of CmKAO were similar in MT and MT+EV but was significantly high (P<0.01) in MT+KAO. High level of KAO transcript was detected in MT+KAO, by contrast, slight level KAO transcript signal was detected in MT and MT+EV through semi-quantitative RT-PCR analysis.4. By cDNA and DNA sequence alignment of the gibberellin biosynthesis key enzymes ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA20-oxidase1(GA20ox1) and GA3-oxidase1(GA3ox1) between wild type and mutant catkins, the result revealed that A-872, A-1115, T-1150in KO2to G-872, T-1115, C-1150bases substitution in KO3which is one allele of KO, which convert Glu-291, Tyr-372, Tyr-384to Gly-291, Phe-372and His-384, and there was no base mutation in the other allele KOI. Through bioinformatics analysis, KO2and KO3protein molecular weight were58.765and58.651kDa, and the theoretical isoelectric point were8.63and8.75, respectively. There are six differences in the secondary structure between KO2and KO3protein, and KO3a transmembrane region and more than KO2. Southern blot showed that for KO there was one copy in wild catkins,but two in mutant catkins.5. The plant over-expression vector pCAMBIA1304-CmKO1was constructed, and using agrobacterium-mediated transformation system, the short catkins were evenly infected in the primordium formation of the male inflorescence. Catkins of mutant catkin (MT1), mutant catkins that transformed with the pCAMBIA1304empty vector (MT+EV1) and mutant catkins which transformed with the pCAMBIA1304-CmKO1(MT+KO1) all began to appear on PCD30days after infection, but the flower clusters quantities of MT+KO1were more than MT1and MT+EV1. CaMV35S was verified in MT+EV1and MT+KO1, it showed that catkins had been infected by Agrobacterium. Real-time PCR analyses were carried out with MT1, MT+EV1and MT+KO1. The expression of CmKO1was significantly high (P<0.01) in MT+KO1than MT1and MT+EV1which the expressions of CmKO1were similar.