Cloning And Functional Validation Of Genes Involved In The Gibberellin Synthesis Pathway In Dwarf Banana

Banana dwarf mutation is one of the common phenotypic variations in the progeny of banana asexual propagation.Up to now,the regulatory mechanism of banana dwarf mutation has not been clearly understood.Many studies have shown that endogenous gibberellin is one of the important hormones affecting plant height,and the mutation of key enzymes encoding genes in gibberellin biosynthetic pathway will lead to plant dwarfing.To explore the molecular regulation mechanism of the key enzyme genes in the gibberellin biosynthetic pathway in banana dwarfing,the wild type parent and its dwarf mutant of Williams B6(Musa AAA Group Cavendish Williams)were used as experimental materials.The c DNA sequences of GA2 ox,GA3ox and CPS,which were related to the biosynthesis of gibberellin,were cloned by RT-PCR with the primers designed and synthesized according to the published c DNA genetic sequences of the two banana materials.The cloned sequences were sequenced and aligned,and the structure and function of the predicted coding proteins were analyzed.At the same time,the transcription and expression of the cloned gene in the pseudostems,leaves and fruits at different developmental stages of the two banana materials were quantitatively detected and analyzed by real-time quantitative PCR,and the plant expression vector of cloned genes were constructed for genetic transformation to study its function.The main results of the study are as follows:1、Six cDNA sequences corresponding to GA2 ox,GA3ox and CPS genes in Williams B6 dwarf mutant and its wild type parent were cloned.The cloning sequences of GA2 ox,GA3ox and CPS genes from dwarf mutant banana were named GA2ox-A,GA3ox-A and CPS-A,respectively.The cloning sequences of GA2 ox,GA3ox and CPS genes from wild type parent banana were named GA2ox-G,GA3ox-G and CPS-G,respectively.The sequence length of GA2ox-A and GA2ox-G were 994 bp,and the open reading frames were 813 bp,encoding 270 amino acids.The amino acid sequences of the two groups were 99.63% similar,and there was a difference of amino acid residues between the two groups.The sequence length of GA3ox-A and GA3ox-G genes were 1096 bp,and the open reading frames were 864 bp,encoding 287 amino acids.The amino acid sequences of the two groups were 98.26% similar,and there were 5different amino acid residues between the two groups.The full length of CPS-A and CPS-G open reading frames were 2163 bp,encoding 720 amino acids.The amino acid sequences of the two groups were 98.89%similar,and there were 8 amino acid residues differences between the two groups.2、The expression levels of GA2 ox in the leaves,pseudostems and fruits of the dwarf mutant were higher than those of the wild type,especially in the 15 and 20 leaf stage,the expression levels of GA2 ox in the leaves and pseudostems of the dwarf mutant were 4.3-28.9 times higher than those of the wild type;The expression level of GA3 ox in the pseudostems of dwarf mutant was lower than that of the wild type,and the expression level of GA3 ox in the pseudostems of dwarf mutant was2.2-32 times lower than that of the wild type.However,in the fruit,the GA3 ox expression level was significantly higher in the dwarf mutant than in the wild type;The CPS gene was lower in both dwarf mutant leaves and pseudostems than in wild type.However,in fruit,CPS was higher in the dwarf mutant than in wild type.3、The molecular weight of GA2ox-A and GA2ox-G proteins were30067.30 Da and 30085.33 Da,respectively,and their theoretical isoelectric points were 5.05 and 5.04.The molecular weights of GA3ox-A and GA3ox-G proteins were 30661.14 Da and 30735.28 Da,respectively,and their theoretical isoelectric points were 9.78.The molecular weights of CPS-A and CPS-G proteins were 82412.15 Da and 82359.00 Da,respectively,and the theoretical isoelectric points were 6.17 and 6.03,respectively.The results of GRAVY analysis showed that the GA2 ox,GA3ox and CPS proteins of dwarfing mutants and wild-type parents were all hydrophilic proteins,and they were all unstable proteins without signal peptides and transmembrane structures.The results of secondary structure prediction showed that the dwarfing mutant and the wild-type parents GA2 ox,GA3ox and CPS proteins contained four conformations including α helix,β folding,extended chain and random curl.Subcellular localization prediction showed that GA3 ox protein of dwarfing mutant and wild-type parent was mainly located in mitochondria,while GA2 ox and CPS protein were mainly located in cytoplasm.The results of conserved domain analysis showed that the dwarfing mutant and the wild type parents GA2 ox,GA3ox and CPS proteins all possessed typical conserved domain of this gene family and were consistent with the characteristics of this gene family.4 、 Plant expression vectors of GA2ox-A gene were constructed.Colony PCR and sequencing results verified the sequence accuracy of cloned genes in the constructed plant expression vector,transformed tobacco with GA2ox-A plant expression vector,and obtained 10 transgenic regenerated tobacco plants of GA2ox-A.In conclusion,the results of this study revealed the structural differences of GA2 ox,GA3ox and CPS,three key enzyme genes in the GA synthesis pathway of dwarfing mutants and their wild type parents.The expression levels of GA2 ox,GA3ox and CPS genes were significantly different in leaves,pseudostems and fruit fingers of the dwarfing mutant and its wild type parents at different developmental stages.The results of this study play an important role in revealing the internal mechanism of banana dwarf mutation.