| Coix Lacryma-Jobi L.is an annual or perennial grass coix and is a monocotyledonous plant.Studies have shown that coix is extremely rich in nutrients,so it has been gradually paid attention to and widely used in nutrition and health care.But the traditional coix in China has the problems of high plant and easy lodging,which leads to the decrease of unit yield.Gibberellin is 2-oxidase(GA2ox)which one of the key enzymes in the process of anabolism,this enzyme by 2-β hydroxylation effect on the activity of gibberellin irreversible lost,,so when the gene expression in plant body,GA3 content and decrease in plants and plant phenotypic shape affected,especially the dwarf plant height general performance characteristics.In this study,the function of GA2 oxidase gene in coix was analyzed in order to optimize plant phenotypic traits and to cultivate dwarf or semi-dwarf plants.The results of this study are as follows:1.Bioinformatics analysis of ClGA2ox2 geneCoix Lacryma-Jobi L(coix).gene sequence was cloned by RACE.The length of the gene sequence was 933 bp,encoding 310 amino acids.Relevant bioinformatics analysis websites or software were used for systematic analysis of the gene sequence.The theoretical relative molecular weight(MW)of the protein is 34.58 k Da,and the molecular formula is C1541H2383N423O458S13.The theoretical isoelectric point(p I)of protein is 5.46.The instability coefficient is 43.97,and the instability coefficient is greater than 40,indicating that the protein might be unstable.The specific functional site of DIOX-N and 2OG-Fell＿oxy of the GA2 ox gene family existed in the amino acids of this gene,and the gene is identified as the GA2 oxidase gene of coix.In addition,coix’s GA2 ox amino acid sequence showed no transmembrane site,no protein signal peptide sequence,and no hydrophobic hydrophilic protein.The secondary structure showed irregular curl and α helix structure,and the tertiary structure was basically consistent with the secondary structure analysis.The sequence homology analysis of the protein amino acid sequences of coix is found to be more than 90% similar to that of sorghum and maize,indicating that the coix is most closely related to sorghum and maize than other plants,which provided a theoretical basis for the classification of coix as gramineae monocotyledons.Based on the above characteristics,the gene was named ClGA2ox2.2.Analysis of partial characteristics of transgenic tobacco with ClGA2ox2 geneThe plant overexpression vector PM1300-35S-ClGA2ox2 was constructed,and tobacco was genetically transformed by leaf disc method.The overexpression transgenic plants(TP)were analyzed,and the differences were not significant compared with wild-type(WT)and empty vector(EV)tobacco.The expression levels of ClGA2ox2 gene in overexpressed transgenic plants TP1～TP3 is 2.82,2.91 and 3.33 times respectively that of the TP in the stem,and 2.56,2.48 and 2.42 times respectively that of the WT in the leaves,respectively,and the differences are extremely significant compared with that of the WT and the EV.That is,the expression levels of this gene in tobacco stem and leaf are significantly increased;The average plant height is16.33±0.12 cm for WTe and 16.23±0.12 cm for EV.The plant height of TP1～TP3 is8.43±0.09 cm,8.13±0.07 cm and 6.33±0.09 cm,respectively.The plant height of TP1～TP3 is nearly half of that of WT.The average diameters of WT to EV tobacco is2.53±0.13 mm and 2.2±0.12 mm,respectively.The diameters of TP1～TP3 were4.20±0.06,4.07±0.13 and 4.13±0.12 mm,respectively.The plant height of TP is nearly twice that of WT tobacco.As the number of stem segments of WT is not significantly different from that of WT tobacco and EV tobacco,it was proved that the height dwarfing of transgenic tobacco was realized by shortening the distance between tobacco stems.In addition,the total chlorophyll content of WT and EV tobacco is 1.81±0.07 and 1.73±0.06 mg/g,respectively,and the content of overexpressed transgenic tobacco TP1～TP3 is 2.19±0.02,2.15±0.08 and 2.29±0.02 mg/g,respectively.Total leaf green increased by 18.78% ～ 28.51% in transgenic tobacco.Therefore,when the gene is overexpressed in tobacco,plant height decrease,stem diameter increase,and chlorophyll content increase significantly.3.Analysis of related characteristics of transgene coix of ClGA2ox2 geneThe plant overexpression vector PM1301-Ubi-ClGA2ox2 and interference expression vector PM1301-Ubi-ClGA2ox2 i were constructed and transformed into coix by stem tip.The results showed that: The expression of ClGA2ox2 gene in interference plant(IP)is 0.68 times that of the wild-type plants(WT),nearly half of that.The TP1～TP4 is 2.87,2.80,2.66,2.02 times respectively that of the WT,respectively.That is,the expression of ClGA2ox2 gene in transgenic plants increased significantly;The plant height is 149.0±2.08 cm in the WT,152.00±1.00 cm in the EV,and 160.00±1.20 cm in the IP.The plant heights of TP1～TP4 is 83.00±1.73,94.33±2.33,112.33±1.45 and 124.50±1.25 cm,respectively.The plant height of the TP is lower than that of the WT,the EV and the IP,and the height of the IP is lower than that of the noEV.It Is found that the seed number of the interference-expressing coix.In addition,The total chlorophyll content of WT and EV tobacco is 1.81±0.07 and 1.73±0.06 mg/g,respectively.The content of overexpressed transgenic tobacco TP1～TP3 is 2.19±0.02,2.15±0.08 and 2.29±0.02 mg/g,respectively.Total leaf green increased by 18.78% ～28.51% in transgenic tobacco.In conclusion,overexpression of ClGA2ox2 in coix can dwarf plant height and increase total chlorophyll content.Conversely,interference of ClGA2ox2 gene expression in coix induces an increase in plant height.These studies provided a theoretical basis for optimizing coix plants to improve crop yield and preserve high-quality germplasm resources. |
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