Effects Of Overexpression Of ClGA2ox Gene On Growth And Development Of Coix Lacryma-jobi L

Gibberellin is a type of plant hormone that plays an important role in regulating the growth and development of higher plants.It is involved in the process of plant seed germination,stem elongation,and fruit ripening.GA2 oxidase is a key enzyme in the gibberellin synthesis pathway.The regulation of active gibberellin levels in higher plants is affected by this enzyme.It can convert active gibberellin into inactive gibberellin.Then achieve the regulation of plant height.Coix lacryma-jobi L.is a medicinal and food homologous crop of one year or perennial grasses,which has high nutrition and medicinal value.However,it is easy to fall down,resulting in low quality and yield,which is not conducive to the preservation of high-quality germplasm resources.At present,there are many studies on GA2-oxidase in other plants,but the function of the GA2 oxidase gene in the gramineous plant Coix lacryma-jobi L has not been reported.This study intends to genetically transform Coix lacryma-jobi L and tobacco with the gibberellin oxidase GA2-oxidase(Cl GA2ox)gene,and further explore the effects of ClGA2ox gene on the growth and development of Coix lacryma-jobi L through analysis and research on transformed plants.It provides research materials and scientific basis for the improvement of plant height,and plays an important role in promoting the breeding process of dwarfing.The results are as follows:1.Bioinformatics characteristics of ClGA2ox geneBioinformatics projections analysis shows:The ClGA2ox gene encodes 333 amino acid,the predicted protein molecular weight is 37.679 k D,and its theoretical isoelectric point is 6.91.The instability coefficient is 35.09,and the protein encoded by ClGA2ox is a stable protein,The sequence includes two conserved domains DIOX?N and 2OG-Fell?Oxy,Homologous alignment shows that it shares 90% of amino acid identities with GA2 ox from other plants in Gen Bank,The phylogenetic tree constructed on the basis of amino acid sequences of GA2 ox suggests that the relationship between Zea mays L.and Sorghum bicolor L.are the most intimate.2.Selection of Reference Genes for Quantitative Real-Time PCR in Coix lacryma-jobi L.Six common internal reference genes were selected as candidate internal reference genes based on the Coix lacryma-jobi L.transcriptome database.The stability assessment software ge Norm and Norm Find were used in combination with the q RT-PCR method to select the internal reference gene of Xingren white-shelled pheasant from six candidate internal reference genes as TUB.3.function Verification of ClGA2ox gene on Coix lacryma-jobi L.In order to study the function of the Coix lacryma-jobi L ClGA2ox gene,this study constructed a PU1301-ubi-ClGA2ox plant overexpression vector,and used the Agrobacterium-mediated stem tip genetic transformation method to introduce the overexpression vector into Xingren small white shell Coix lacryma-jobi L.A total of14 T0 transgenic Coix lacryma-jobi L plants with hygromycin resistance were obtained.The measurement results of related indexes of transgenic Coix lacryma-jobi L plants showed that,compared with wild-type Coix lacryma-jobi L plants,the expression of ClGA2ox transgenic coix plants increased and the plant height decreased to varying degrees(31.21 cm lower than the average plant height of wild type),and the chlorophyll content increased.In order to further study the function of Coix lacryma-jobi L ClGA2ox gene,at the same time,the Coix lacryma-jobi L ClGA2ox gene was transformed into tobacco,and a total of 36 Cl GA2ox transgenic tobacco plants were obtained.Five plants were randomly selected from them for relevant research.The results of fluorescent quantitative PCRshowed that the expression of the target gene was not detected in wild-type tobacco,while the expression of the target gene in transgenic tobacco plants was significantly higher than that in the control group.Tobacco plants showed dwarfing traits,the leaves became smaller,the internode distance became shorter,and the chlorophyll content increased.These results show that the overexpression of Coix lacryma-jobi L ClGA2ox gene in tobacco can also achieve the same function as Coix lacryma-jobi L,which can cause the transgenic tobacco dwarf phenotype.