Effects Of Environmental Stress On Growth,metabolism And Gene Expression Of Terpenoid Enzymes In Azadirachta Indica

Azadirachta indica(A.indica)was recognized as the most ideal biological and insecticidal plant in the world,azadirachtin is the main active component.Although it has been introduced into our country more than 30 years,there is still lack the theory and technolog of precise cultivation of it.In order to construct and improve the theoretical system of efficient cultivation of A.indica as soon as possible,and further tap the production potential of azadirachtin,we selected two most concerned environmental factors,water and light,as breakthrough point.The young seedlings and adult clones of A.indica were used as the experimental materials to analyze the growth of A.indica seedlings and the response of its primary metaboli enzymes on different water and light stress,and explore the formation and accumulation level at the vegetative growth stage of young age under environmental stress.Meanwhile,the content of azadirachtin,the activity of key enzyme in its synthesis pathway and the expression of its coding gene were measured simultaneously under the condition of drought stress in the reproductive growth stage,in an attempt reveal the environmental induction mechanism of azadirachtin formation and accumulation from levels of end product,protein and nucleic acid.The main findings are summarized as follows:(1)The condition of natural light and field capacity of 60%-65%(W₀L₀)most favorable for the growth of A.indica seedlings in the vegetative growth stage,and the growth rate greater than other treatments by 9.7%-294.9%for seedling height,28.4%-173.0%for diameter and 34.7%-569.4%for biomass,respectively.It shows that water and light stress could inhibit the growth.Under the condition of drought stress,A.indica can maintain a smaller variation in seedling height,ground diameter and biomass by regulating the activity of enzymes in carbon-nitrogen metabolism such as Sucro synthase(SS),Sucrose phosphate synthase(SPS),Nitrate reductase(NR),glutamine synthetase(GS),glutamate synthase(GOGAT),and accelerating the accumulate of soluble sugars,soluble proteins and free prolines,which showed a greater drought adaptation in growth stage of seedling.Azadirachtin can be synthesized and accumulated in the leaves of seedling at the stage of vegetative growth,and the content is the highest under the conditions of natural light and field water capacity of 40%-45%(W₁L₀),the content of azadirachtin A,azadirachtin B and azadirachtin A+B is 0.014%,0.0026%and 0.017%respectively,it is greater than W₀L₀by 40.00%,8.33%and 33.87%,respectively.It shows that shading or excessive drought stress are not conducive to the synthesis of azadirachtin in leaves.(2)In the reproductive growth stage of A.indica,drought stress increased the 1000 kernel weight and kernel proportion by 7.8%and 8.9%,respectively,and contribute 20%to the accumulation of azadirachtin,On the contrary,the growth rate of seeds was accelerated by sufficient soil water supply,but the organic matter content in the seeds did not increased synchronously.light stress lead to the decreased of the 1000 kernel weight and content of azadirachtin by 2.7%and 1.4%.The correlation analysis showed that the content of soluble sugar and protein was positively correlated with azadirachtin in the early stage of seed development,while both were negatively correlated with azadirachtin in the middle and late stages.(3)The activity of terpenoid synthase was higher under drought stress in the late development stage of A.indica seeds.In the early stage of seed development,the catalytic enzymes of HMGS,HMGR and DXS have higher biological activity,as the seed matures,it gradually decreases,while the catalytic enzymes of SS,SQLE still have higher biological activity,and the activity was the highest under drought stress.(4)The transcripts of twelve samples of A.indica kernel(kernel formation and near ripening)were sequenced using the Illumina high-throughput sequencing technique in this study,and acquired huge numbers of gene sequences information.The number of clean Reads obtained by high-throughput sequencing accounted for over 98.9%of the number of raw Reads,and the sequencing base quality value Q₃₀was all over 92.9%,indicating reliable sequencing quality.Then de novo assembly of the clean reads by Trinity,acquiring 40120 Unigenes,a total of 23372 Unigenes were functionally annotated.In W₁-a-VS-W₂-a and W₁-b-VS-W₂-b groups,the genes with more than a twofold difference in the expression quantity were defined as the differentially expressed genes(DEGs),The results of GO and KEGG enrichment analysis showed that DEGs related to terpenoid synthesis were not enriched in the fruit nucleus formation stage,two DEGs were enriched in the near ripening stage of fruit.After WGCNA analyses,there haven't found any genes enriched to the pathway of terpenoids synthesis,which was in the module significantly correlated to azadirachtin.48 Unigenes were involved in azadirachtin biosynthesis in A.indica kernel.genetic expression were up-regulated in W₂at the nearly ripe fruit period,and 11 of them could be used as candidate genes for Azadirachtin synthesis,genetic expression were conformed with the activity of catalytic enzymes of HMGR,HMGS,DXS,SS,SQLE,and basically agreed with the variation of inzadirachtin content.The reasons for the increase of azadirachtin content in kernel caused by drought were explained from many aspects,which provides the basis for the artificial regulation of azadirachtin,and lay a theoretical foundation for further study on the effects of environment on the metabolism of A.indica.