Screening Of High EGCG3"Me Tea Germplasm Resources And Study On Influencing Factors Of Methyltransferase Related Gene Expression

(-)-Epigallocatechin-3-O-（3-O-methyl）gallate（EGCG3"Me）has multiple health functions,but the content of which was quite low in tea plant.Factors,such as cultivars,growing environment,maturity,picking time and season,and processing methods etc.can affect its content in tea plants,while the mechanism of the EGCG3"Me formation is not clear yet.After screened the 84 innovative tea germplasms,the distribution of EGCG3"Me in different germplasms,shading treatments and withering processes was investigated.The expression of methylated genes like Cs CCo AOMT1、Cs CCo AOMT2、Cs COMT1、Cs COMT2 were measured and analyzed with the data of EGCG3"Me investigation to study the mechanism of the methylation of EGCG in tea plant.（1）The content of EGCG3"Me on different leaf position in 84germplasms were detected and classified into 3 groups（high content,middle content and low content）by the results of clustering analysis.The average content range of five leaf positions in high content group which has 13 germplasm was 6.27mg/g～17.47mg/g.The content range from bud to the 4^thleaf in the highest germplasm Phoenix single bush was13.81-23.22mg/g.Middle content group has 40 germplasms of which content range of 5 leaves was 2.71mg/g～6.15mg/g.Low content group has 31 germplasms of which content of 5 leaves ranged 1.56mg/g～2.58mg/g.（2）The investigation of the content of 5 buds and leaves of the 31innovative germplasm in spring,summer and autumn all showed that it increased with the growth and from spring,autumn to summer.In spring,the diversity of the content in different leaf position and in high content germplasm was more significant.（3）3’RACE technique was employed to get the 3’UTR of 7Cs COMT sequences（from the genome data of Suchazao）due to their high homology,and 2 of them were obtained for further q PCR test.The others cannot be cloned in the leaves may attributed to the tissue-specific expression.The expression of 2 Cs CCo AOMT in tea was verified,and 2specific fluorescent quantitative primers were obtained.（4）There was no significant correlation between EGCG3"Me content and EGCG content among different germplasms,but it was related to the gene expression of Cs COMT1,Cs COMT2 and Cs CCo AOMT2.The content of EGCG3"Me in 3^rdleaf position of Jinmudan was significantly high than that of Jinguayin,which was4.32mg/g and 1.06mg/g,respectively.However,the content of EGCG in the same position in these two cultivars showed no difference,which was65.36mg/g and 61.33mg/g.In one bud two leaves,the EGCG3"Me content of Y21 was 6.58mg/g and EGCG content was 137.82mg/g.But Fudingdahao which was no EGCG3"Me detected did not follow the pattern,of which the EGCG content was 120.01mg/g.The expression pattern of Cs COMT and Cs CCo AOMT was opposite in the 3^rdleaf.The Cs COMT expressed higher in Jinguayin than in Jinmudan,while the Cs CCo AOMT expressed reversely.The expression pattern of Cs COMT and Cs CCo AOMT1 of one bud two leaves was Fudingdahao>Y21,and that of CCo AOMT2 was Y21>Fudingdahao.The absence of EGCG3"Me content in Fudingdahao may be related to the high expression of Cs COMT2.（5）The distribution of the EGCG3"Me content in these germplasms and treatments were the same,which increased accompanying with the leaf maturation,stimulated by a six-days shading treatment,and the high content germplasms responded faster.EGCG3"Me in Jinguayin under normal growth increased 62.37%（compared with day zero）,and increased 88.73%after shading for 6 days.In Jinmudan,it increased44.82%compared with the day zero（4.32mg/g）after normal grow under nature environment,and stimulated 70.85%after six-days shading.（6）There is no pattern for the EGCG3"Me distribution of germplasms under withering.But withering can stimulate the EGCG3"Me generation in tea leaves,which got peak at 24hr of process.But there is no rule for the expression of the transmethylases after 42hrs of withering.The highest content of EGCG3"Me in Y21 was 11.48mg/g.The increased expression of Cs COMT and Cs CCo AOMT1 enhance the EGCG3"Me generation in high content germplasm,which showed the withering time should be under 24 hours.In Y21 germplasm,the content variation within 42 hours mainly caused by the change of the EGCG content and the Cs COMT expression.The reason is that maybe the plant altered its distribution model to adapt to the stress,which lowered the content of EGCG and the Cs COMT had to involved into the lignin mechanism to compensate the methylation of Co A.That is why the EGCG3"Me maintained a stable level after 42 hours withering.The expression of Cs CCo AOMT only accompanied with the change of EGCG3"Me within the first 6 hours in withering process.