| In recent years, the isolation and identification of volatile terpenoids synthase genes andtheir expression regulation and function characteristics have made great progress, also,terpenoids metabolic engineering has attracted widespread attention. But until now, onlyseveral kinds of enzymes have been studied in detail.α-Farnesene synthase is the final enzyme in the sesquiterpere α-farnesene synthesispathway. Until now, studies on α-farnesene synthase mainly focus on the mechanism ofsuperficial scald production and its relationship with α-farnesene synthase gene expression.Overexpression of α-farnesene synthase in plants, which can influence the production of otherterpenes and plant biological properties, has not been reported.In this study, we studied the difference of growth characters between transgenic plantwhich overexpressed α-farnesene synthase gene from apple tree (Malus×domestica) with35Spromter and wild-type tobaccos, and their molecular mechanisms using homozygous T3generation of transgenic tobacco (NC89). We also isolated the promoter sequence ofα-farnesene synthase gene on genomic DNA from apple by TAIL-PCR method andpreliminarily analyzed its functions. The results will contribute to studies on the influence ofsesquiterpene metabolism engineering on the plant growth and the factors of α-farnesenesynthase gene regulation. The main results and conclusions in this thesis are presented asfollows:(1) α-AFS overexpression has affected the growth characteristics of transgenic tobaccoand hormone levelsT3generation homozygotes of transgenic and wild type tobacco were cultivated in sameconditions. There was no significant difference in morphology after the early30days betweentransgenic and wild-type plants. However, the transgenic plants growth fast after45days, andplant morphology such as plant height, stem internodes, leaf number between transgenic andwild type was significant in difference until60days later, and the flower buds emerging andflowering time of transgenic plant was50days earlier than that of wild type plants.We measured the GAs, ABA and IAA contents at the4leaves,8leaves,12leaves,14leaves and16leaves growth stages by ELISA, and found that in the early development, the hormone levels did not show significant difference in the transgenic and wild-type plants, butin the bud and flowering period, the GAs, ABA and IAA contents in transgenic plants were102.28%,109.50%,257.37%(buds), and115.75%,223.53%and289.64%(blooming) higherthan those in wild-type, which indicated that overexpression of α-AFS gene changed thehormone levels, and then influenced the plant height, flowering time characteristics oftransgenic plants.(2) α-AFS overexpression affected the expression levels of key enzymes in MVA and MEPpathways and the flowering-related genes in transgenic tobaccoWe estimated the transcription levels of the key enzymes NtHMGR2, NtFPS, NtDXS andNtDXR of MVA and MEP pathway by qPCR. The results showed that overexpression ofα-AFS improved the expression level of NtHMGR2gradually from seedling to floweringperiod, while the expression of NtFPS was lower at4and8leaves stages and higher at12leaves stage than wild-type, and then decreased until the flowering time. Transgenic plantsexhibited higher NtDXS expression level from4to8leaf stage, but lower level than the wildtype after8leaf stage, showing a downward trend. NtDXR expression level of transgenic plantwas slightly higher than that of wild-type only at4leaves stage, and was lower at other fourstages.GAs is the downstream product of MEP pathway. The expression levels of the enzymegenes related to the GAs synthesis were analysized by qPCR, the results showed that theNtGA20ox1expression level improved gradually with the development, and at the12leavesstage, the expression levels in all three transgenic plants were4.87,2.40and2.46times towild-type, respectively. Although the NtGA20ox1expression level decreased in budding andflowering period, it was still higher than that in wild-type plants. The expression levels ofNtGA20ox2increased markedly in transgenic plants at the bud and flowering period,especially the T3-3and T3-5strains. NtGA2ox1, NtGA2ox2, NtGA2ox3and NtGA2ox5showedhigher expression level in seedling stage, and then began to decline until flowering.The expression pattern of flowering-related genes in tobacco were detected bysemi-quantitative PCR, the results showed that the expression of NtMADS4in T3-1, T3-3andT3-5and NtMADS11in T3-1, T3-5were all significantly earlier than those in wild-type plants,but T3-3line didn’t show significant difference in expression level comparws to wild-typeplants.(3) α-AFS overexpression affected the synthesis of other terpenoids, alkaloid and sterols intransgenic tobacco.The extractives of transgenic and wild-type lines were analysed by GC-MS, and the results showed that no α-farnesene was detected in all lines, but in transgenic plants, the level ofβ-caryophyllene was increased6fold, trace caryophyllene oxide, andrographolide, and a newditerpene sclareol were also detected. HPLC analysis showed compounds levels were variousbetween transgenic and wild-type tobacco, and what kinds of those compounds are stillunknown.We also found that the alkaloid content of dry leaves was higher but the content of totalsterols was lower in the transgenic plant than the wild-type.(4) α-AFS overexpression improved the stress resistance of transgenic tobaccoUnder treatment with natural senescence, MV, NaCl,45C, compared to wild-type, thenatural senescence resistance ability of leaf discs from transgenic tobacco was higher, butmore sensitive to20μM MV stress, the seed germination rate was no visible difference underNaCl stress, but the seedlings of transgenic plants grew better on the MS mediumsupplemented with100mM NaCl, but there was no obvious difference between them with150mM NaCl stress. Under45C stress, the Pn decreased markedly in the transgenic plantscompared with25C, but the value was similar to the wild type, and had lower relativeconductivity and injury degree than wild-type.(5) The isolation and function analysis of the α-AFS gene promoterWe isolated the α-AFS gene promoter sequence from the ‘white winter pearmain’ apple（Malus x domestica）by TAIL-PCR method, and the online analysis showed that thesequence has many obvious TATA box and CAAT box, the element associated with hormone,and HSE element involved in thermal stress reaction, anaerobic induced element and otherimportant cis-acting elements.The promoter sequence fragment was inserted into the upstream of GUS reporter gene andintroduced into tobacco. GUS activity analysis showed that the promoter can induce GUSgene expression, and the expression activity was different in various length of promotersequence. We treated the transgenic tobacco, which was transformed with the whole promotersequence, with GA, ABA and SA, MeJA, ethephon (ethephon) and45C heat shock, and theresults showed that GA, MeJA, ethephon treatment enhanced the GUS activity, but the SAand ABA treatment exhibited reverse results. Heat shock treatment improved GUS activitywhen promoter sequence contained the HSE element (sequence2-1), but didn’t for otherseuquences. |
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