| Stevia rebaudiana Bertoni is a high-profile sweet herb,because its leaves can extract steviol glycosides which with the characteristics of high sweetness(250?450 times more than sucrose)and low calories(only 1/300 of sucrose).Steviol glycosides commonly known as stevia sugar,it is the most recommended natural calorie-free sweetener,which is known as "the best natural sweeteners" internationally.In addition to being a sweetener,more and more scientific researches have indicated that steviol glycosides can prevent and treat obesity,diabetes,hypertension,hyperglycemia,cavities,and have medicinal properties such as anti-inflammatory,antioxidant,antibacterial,antitumor and enhance immunity.Stevia leaves can accumulate high concentrations(up to 30%)of more than thirty different glycosides derived from the tetracyclic diterpenesteviol.These glycosides have the same aglycone steviol,but they have differet sweetness,flavor and biological activity,besause the type and quantity of glycosyls which connected to steviol were different.In these steviol glycosides,the two main glycosides are stevioside(ST)and rebaudioside A(RA),they traditionally make up the majority of the sweetener(60?80%of the total glycosides content).ST has intense sweetness(250?300 times sweeter than sucrose)and the potential medicinal values;RA has more intense sweetness(350?450 times sweeter than sucrose)and most desirable flavour profile close to sucrose.So in order to meet the different needs,it is one of the goals of stevia breeding to increasing the conten of single glucoside component.This thesis includes two parts contents:1.A rare low-RA mutation plant was found through this experimentation.Then the causes of extremely low RA content of the optimizing plant were proved from the levels of gene and protein of SrUGT76G1,a key gene of steviol glycosides synthesis.2.The gene expression features of fifteen genes involved in SGs biosynthesis were studied by semiquantitative RT-PCR analysis,SrUGT76Gl were analyzed by fluorescent quantitative PCR.Moreover,through the promoter cloning and sequence analysis,the expression regulation mechanism of SrUGT76Gl gene was preliminary understood.The main research contents and results are as follows:1.In the present study,the concentration of stevioside and rebaudioside A in stevia leaves of two stevia cultivars 'zhongshan No.3','zhongshan No.4'(Z03 and Z04 for short,respectively)and their 37 F1 hybrids offspring were analysised by HPLC.The results show that the content of ST range changes from 1.18%to 9.83%;the content of RA range changes from 5.15%to 15.36%;the total content of ST and RA range changes from 10.26%to 19.67%;and the rate of RA to ST and RA total content range changes from 34%to 90.42%,they are all obey normal distribution.In the samples,a offspring,named 011 had a character of low-RA,its ST content was 15.9%,and its RA content was just 0.4%.This is rare in about 500 samples of other hybrid progenies and stevia germplasm resources collected from home and abroad.Then 011 was entitled 'zhongshan No.5',Z05 for short.2.In previous studies of steviol glycosides biosynthes,UGT76G1,a kind of uridinediphosphate-dependent glycosyl transferase,which catalyzed the glucosylation of the C-3' of the glucose at the C-13 position,is responsible for the convertion from ST to RA,so mutation identification was done by sequencing the candidate gene,SrUGT76G1.In this study molecular analysis of two varieties revealed a heterozygotic nonsense mutation of c.389T>G(p.L121X)in SrUGT76Gl.Meanwhile,we found some amino acid substitutions significant change the protein structure.And the difference of enzyme activity in vitro between two varieties proved the lack of functionality of SrUGT76G1 of Z05.So the nonsense mutation and amino acid substitution mutation resulted in the low levels of Rebaudioside A.3.In the meantime of studying SrUGT76G1 gene expression features,fifteen genes involved in SGs biosynthesis of S.rebaudiana were examined by semiquantitative RT-PCR analysis.In this study,the influences of different temperatures(15?,25?,35?),dehydration,different photoperiods,and different growing stages on the changes of steviol glycosides contents and transcription levels of fifteen genes were examined using HPLC and RT-PCR,respectively.The observations showed that the transcript levels of all the fifteen genes were maximum under 25?treatment,and the transcription of SrDXS,SrDXR,SrMCT,SrCMK,SrMDS,SrHDS,SrHDR,SrIDI,SrGGDPS,SrCPPSl,SrUGT85C2 and SrUGT76G1 were restrained both in low temperature(15?)and high temperature(35?).Most genes in SGs biosynthesis pathway exhibited down-regulation in dehydration.To elucidate the effect of photoperiods,the plants were treated by different simulated photoperiods(8L/16D,10L/14D,14L/10D and 16L/8D,L=Light;D=Dark),but no significant transcription changes were observed.In the study of growing stages,there were evident changes of SGs contents,and the transcript levels of all the fifteen genes were minimal in fast growing period,and exhibited evident increase both in flower-bud appearing stage and flowering stage.4.The relative expression levels of SrUGT76G1 were studied under short-day(8L/16D)and long-day(16L/8D)treatments,in different growing stages and at the different times of the day by fluorescent quantitative PCR.The result shown that it was 7.42 times of the relative expression of SrUGT76G1 under the long-day treatment than that under short-day.The relative expression of SrUGT76G1 in fast growing period,flower-bud appearing stage and flowering stage were 0.41,0.72 and 82.7 times of that in the control.And at the different times of the day,the relative expression of SrUGT76Gl at 8:00,12:00,16:00,20:00 and 24:00 were 1.64,1699.75,69.25,24.94 and 24.60 times of that at 4:00,respectinely.5.In order to understand the regulatory mechanism of SrUGT76G1 gene,the SrUGT76G1 promoter was cloned from S.rebaudiana by high-efficiency thermal asymmetric interlaced PCR(hiTAIL-PCR).As a result,a 2283-bp 5' flanking region was isolated.In silico analysis showed that this sequence contained 475 acting elements which included TATA-box and CAAT-box,MYB binding sites,phytohormone responsive elements,stress responsive elements,tissue-specific expression elements and other elements required for reaction to environmental stimuli.In order to study the promoter function,a 1982-bp promoter sequence of SrUGT76Gl was inserted upstream of the GUS reporter gene replacing the CaMV35S promoter of pCAMBIA1 301-220.The plant expression vectors pCAMBIA1301-220-UGT76G1P and pCAMBIA1301-220 were transformed into Arabidopsis thaliana and S.rebaudiana seedlings respectively by Agrobacterium vacuum infiltration method.The transient expression suggested that SrUGT76G1 promoter could drive the GUS reporter gene to express both in A.thaliana and S.rebaudiana seedlings.To our knowledge,this is the first report about the gene regulatory mechanism of stevia. |
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