| Pueraria lobata is a medicinal and edible plant that is rich in starch and flavonoids and has great medicinal and edible value.In this study,we studied the agronomic characters,leaf photosynthetic parameters,starch accumulation and the change of key enzyme activity of starch synthesis pathway during the development of Pueraria lobata using Guige 1 and Guige 8,that Guige 1 was the main cultivar in Guangxi production area and Guige 8 was a high-yield and high-starch processing type.We cloned the small subunit gene(sA GP)and large subunit gene(LAGP)of Pueraria lobata starch synthesis key enzyme(AGPase)gene,and analyzed the spatio-temporal expression of large and small subunit genes by real-time quantitative PCR technique.The SPSS software was used to analyze the correlation of leaf photosynthetic characteristic parameter,agronomic characters,starch accumulation,starch synthase activity and AGPase gene expression in root tuber during different growth and development stages in Pueraria lobata.Get the following result:1.The dynamic change of agronomic traits during the development of Pueraria lobata.During the whole growth period,the fresh weight,yield,dry matter content and average root length of No.8 were higher than that of No.1,that of No.8 reached a significant difference level in November and December,the highest values of No.8 were 1.9 times,1.9 times,1.58 times and 1.19 times of No.1,respectively.The fresh weight per plant,yield and dry matter content of two kudzu varieties increased slowly from June to August,and increased rapidly from September to December.The average root length of root tuber increased rapidly from June to July,and increased slowly from August to December,and reached its maximum in November and then tended towards stability.The variation trend of average width of root tuber was basically the same,the average width of root tubers increased slowly from June to August,and increased rapidly from September to November,reached its maximum value in November and then tended towards stability,except that the average root diameter of No.8 in June and July was slightly larger than No.1,all the other months showed that the No.1 was higher than No.8,and the average width of root tubers in the two cultivars reached a significant difference level from October to December.2.Dynamic changes of leaf photosynthetic parameters of Pueraria lobata.During the whole growing period,the net photosynthetic rate,stomatal conductance,intercellular CO2 concentration and transpiration rate of Pueraria lobata showed a single peak curve,which reached the peak in October and then decreased rapidly,as a whole the photosynthetic parameters of No.8 was higher than those of No.1,the highest values were 1.28 times,1.23 times,1.01 times and 1.17 times of No.1,respectively.The net photosynthetic rate of No.1 increased slowly from June to August and increased rapidly after August,while that of No.8 increased rapidly after June,and that of the two varieties gradually decreased after October,except that the difference was not significant in June,the difference was significant in the other months.The stomatal conductance of No.1 increased slowly from June to July and gradually increased from August to September,while that of No.8 gradually increased from July to September,the stomatal conductance of both cultivars increased rapidly from September to October and decreased rapidly from October to December,except that there was no significant difference between the two varieties in June and July,there were significant differences in all the other months.The intercellular CO2 concentration of No.l and No.8 increased slowly from June to July,that of No.1 decreased slightly in August and that of No.8 increased gradually in August,both varieties increased rapidly from September to October,and declined rapidly from October to December,except that there was no significant difference between the two varieties in June,July and October,there were significant differences in all the other months.The transpiration rates of the two varieties fluctuated at a certain level from June to September,increased rapidly from September to October and decreased rapidly from October to December,except that there were significant differences between the two varieties in October and November,there was no significant difference in all the other months.3.The dynamic changes of starch accumulation and key enzyme activity of starch synthesis during the development of Pueraria lobata.During the whole gro,Awth period,the amylose content,amylopectin content and total starch content in the roots of two Pueraria lobata cultivars gradually increased,rapidly accumulated from June to October,reached the highest value in October and then tended to stabilize.In each period,amylose content of No.1 was higher than that of No.8,and the highest value was 1.19 times of No.8.The amylopectin content and total starch content of No.8 were higher than No.1,and the highest values of No.8 were 1.21 times and 1.08 times of No.1,respectively.The amylose and amylopectin contents of the two cultivars were not significantly different in June,there were significant differences from July to December.The total starch contents between the two varieties were not significantly different from June to July,and there were significant differences from August to December.The activity of the key enzyme of starch synthesis which included AGPase,SSS,GBSS and SBE,showed a single peak curve during the whole growth period,and reached its peak value in October,as a whole the activity of the key enzyme of No.8 was higher than that of No.1,the highest values of No.8 were 1.31 times,1.35 times,1.29 times and 1.1 times of No.1,respectively.There was no significant difference in AGPase activity between the two cultivars in June and December,there were significant differences from July to November.There was a significant difference in SSS activity between the two cultivars in September and October,there was no significant difference in other months.There was no significant difference in GBSS activity between the two cultivars from June to August,there was a significant difference from September to December.There was no significant difference in SBE activity between the two cultivars in June,there was a significant difference from July to December.4.Cloning of large and small subunit genes of AGPase,a key enzyme for starch synthesis in Pueraria lobata.Using PCR homologous cloning technology,the small subunit sAGP gene cDNA fragment 1679bp of Pu.eraria lobata AGPase gene was cloned,encoding 463 amino acids,the nucleotide sequence homology of sAGP gene of Pueraria lobata,Phaseolus vulgaris,Pisum sativum Linn,Cicer arietinum,Lens culinaris was more than 90%,the amino acid sequence homology was more than 94%,which is highly conserved.The amino acid sequence homology between Pueraria lobata sAGP and Phaseolus vulgaris PvAGPS1 was as high as 96%,and they are clustered into one class by cluster analysis of phylogenetic trees,while they had the closest genetic relationship.The large subunit LAGP gene cDNA fragment 1237bp of Pueraria lobata was cloned,encoding 307 amino acids.The nucleotide sequence homology of LAGP gene was 77%～93%among Pueraria lobata,Phaseolus vulgaris,Pisum sativum Linn etc,the amino acid homology was 69%-88%,the LAGP gene homology of Pueraria lobata and Phaseolus vulgaris was the highest,the nucleotide sequence homology was up to 93%,the amino acid homology was up to 88%.5.Dynamics of spatio-temporal expression in large and small subunit genes of AGPase during the development of Pueraria lobata.The expression of sAGP gene and LAGP gene in root tuber and root hills of the two varieties was at a low level from June to September,that was in a high level from October to December,reached a significant level in root tuber,and that of No.8 was higher than No.1 in root tuber during the whole development period in general.The expression of sAGP gene in root tuber of the two varieties showed a bimodal trend,the peak value of No.1 was in August and October,and the peak of No.8 was in July and October,except that there was no significant difference between the two varieties in June,there were significant differences in all the other months.The expression of LAGP gene in root tuber of No.1 decreased gradually from June to August,and recreased gradually from September to December,the expression of No.8 was a single peak curve.In root hills,the expression change of sAGP gene and LAGP gene was roughly the same,that is,the two genes expression change showed that No.1 was slightly higher than No.8 in June and October,and the difference was not significant,the No.8 is slightly higher than No.1 in September,but the difference was not significant,No.1 was significantly higher than No.8 in August and December,the differences were different only in July and November,there was a significant difference in sAGP gene expression of the two varieties,there was no significant difference in the expression of LAGP gene,No.8 was slightly higher than No.1 in July,No.1 was higher than No.8 in November.In the stem,the expression change of sAGP gene and LAGP gene was roughly the same,that is,both gene expression of two varieties were extremely low during September and October,the expression level was high in other months,both gene expression of No.1 were higher than No.8 in July and August except for June,No.8 were higher than No.1 from September to December,the differences between the two varieties reached significant levels in July,August,November and December,while the difference was not significant in the the other months.In the leaf,the expression of sAGP gene in the two cultivars was generally at a high level from June to September,that was in a low level from October to December.The LAGP gene of the two cultivars was at a low level from June to October,that was in a high level from November to December.The differential expression change of sAGP gene and LAGP gene in two varieties were generally consistent,that is,except for June and July,No.1 was significantly higher than No.8 from August to September,and No.8 was slightly higher than No.1 between October and November,but the difference was not significant.The sA GP gene and LA GP gene of two Pueraria lobata cultivars had tissue specificity at each stage,when the expression of root tuber reached the maximum in October,the expression levels of sAGP and LAGP genes were as follows:root tuber>root hill>stem>leaf.The sAGP gene of No.1 was generally highly expressed in stems and leaves from June to September,and that was highly expressed in root tubers and root hills from October to December,the expression level of No.8 was high in root tuber during the whole development period.The expression of LAGP gene in different parts of the two cultivars was low from June to September,and it was high from October to December.6.Correlation analysis.The net photosynthetic rate of leaves in Pueraria lobata was highly significant and positively correlated with stomatal conductance,intercellular CO2 concentration,and it was significantly and positively correlated with transpiration rate.The net photosynthetic rate,stomatal conductance and intercellular CO2 concentration were highly significant and positively or significantly positively correlated with amylopectin content and total starch content,and they were positively with amylose content,fresh weight per plant,yield,dry matter content and the average width of root tubers,but the difference was not significant,and the coefficient value was relatively small.Fresh weight per plant,yield and dry matter content were highly significant and positively correlated with amylopectin and total starch content.The amylose content of Pueraria lobata was highly significant and positively correlated with the activity of SSS,and it was significantly and positively correlated with the activity of AGPase,GBSS,SBE and the expression of sAGP gene and LAGP gene in root tuber.The content of amylopectin and total starch were highly significant and positively correlated with the activities of the four key enzymes of starch synthesis,the expression of sAGP gene and LAGP gene in root tuber.The expression of sAGP gene in root tuber was highly significant and positively correlated with the activity of AGPase,SSS,GBSS and SBE.The expression of LAGP gene in root tuber was significantly and positively correlated with the activity of SSS and GBSS,and it was positively correlated with the activity of AGPase and SBE,but the difference was not significant.Fresh weight per plant,yield and dry matter content were highly significant and positively correlated with the expression of LAGP gene in root tuber.The average root length was highly significant and positively correlated with the activity of SBE,it was significantly and positively correlated with the activity of AGPase,SSS and GBSS,the expression of sAGP and LAGP gene in root tuber.The net photosynthetic rate,stomatal conductance and intercellular CO2 concentration were highly significant and positively correlated with the activity of AGPase,SSS,GBSS and SBE and the expression of sAGP gene in root tuber,the intercellular CO2 concentration was significantly and positively correlated with the expression of LAGP gene in root tuber.Transpiration rate was highly significant and positively correlated with the activity of AGPase,SSS and GBSS,it was significantly and positively correlated with the activity of SBE,and it was positively correlated with the expression of AGPase gene in root tuber,but the difference was not significant. |
PDF Full Text Request