Effects of Exogenous Methyl Jasmonate and Gibberellin on Floral Development and Its Implication in the Utilization of Heterosis.pdf

1、Oilseed rape oil is one of the most important vegetable oils for edible consumption inthe world, and the meal with high protein content and well balanced amino acids is avaluable supplement in animal feed mixtures. The e

2、conomic significance of the cropdemands intensive studies on its physiological processes, including its transition fromthe vegetative to the reproductive stage. Delayed flowering time, insufficientflowering period, or ab

3、normal floral organs including male sterility may result in asignificant decline of yield in oilseed rape production. However, male sterility is acrucial element in a heterosis utilization system guarantying a yield prom

4、otion.Flower development involves a large group of genes that respond to environmentalstimuli and endogenous cues. The role of jasmonates and gibberellins in floweringremains largely not understood. The advent of molecul

5、ar biology era provides us anew perspective in comprehending the mechanism of floral organ development andmale sterility. In this paper we investigated the effect of exogenous methyl jasmonate(MeJA) and gibberellins (GA3

6、) on the development of floral development and itsimplication in the utilization of heterosis in oilseed rape (Brassica napus L.). In Chapter 3.1 and 3.2, effects of exogenous MeJA and GA3 on the floweringtime, floral o

7、rgan morphology, and transcript levels of a group of genes implicated infloral development are demonstrated. Through controlled greenhouse experiments, wefound that the effect of MeJA depended on both plant genotype and

8、jasmonate dosage.MeJA promoted maximum flowering when it was applied to the cultivars ofearly-flowering types of oilseed rape. In addition, a concentration of 100 μM resultedin the most number of early open flowers, in c

9、omparison with the results obtained forthe concentrations of 50 μM and 80 μM, indicating a dosage effect. Moreover, theapplication of high concentrations of MeJA (100μM) also produced various kinds ofabnormal flowers. Mo

10、lecular approaches showed that the combined actions of thefloral identity genes, specifically BnAP1, BnAP2, BnAP3, BnAG1, and BnPI3, asreflected by their respective relative transcript levels, were responsible for causin

11、g thedifferent kinds of flower abnormalities previously un-described. On the other hand,the application of 50 μM GA3 gave rise to the shift of flowering time varied from 0.6to 2.6 days among six winter rapeseed cultivars

12、. Some cultivars did not respond toGA3 in terms of flowering time, whereas others (Xinyang-7833, S-205, Mei-Jian andFu-You 4) started to flower significantly earlier after the treatment of GA3 than theirrespective untrea

13、ted control. In addition, the 50 μM GA3 had a clear effect on floweropening and the elongation of filament epidermis cells. Analysis of relative transcriptlevels of a group of functionally identified genes indicated that

14、 GA3 regulated theexpression of the floral homeotic genes such as BnAP3, BnAG1, BnPI3 and the geneson the GA signal transduction pathway like BnRGA2, BnGA1, as well as the genesbelonging to MYB family as BnMYB21, BnMYB24

15、 and BnMYB28. As the disequilibrium of jasmonates or gibberellins results in a variety ofabnormal flowers caused by the combined action of a group of identified genes, thegenome-wide transcriptome changes and the uniden

16、tified GA3- and MeJA responsivegenes were further interested. In Chapter 3.3, we employed cDNA-amplifiedfragment length polymorphism (cDNA-AFLP) analysis to identify genes thatexhibited modulated expression following the

17、 application of MeJA and GA3 to flowerbuds. By using 64 primer pair combinations, about 2787 cDNA fragments werecounted and all bands longer than 50bp in size were compared among the fourtreatments, viz. the water contro

18、l, the 50μM MeJA, the 50 μM GA3, and the 100 μMMeJA. A total of 168 transcript derived fragments (TDFs) were differentiallydisplayed among the treatments. The expression pattern of several of these genes wasconfirmed by

19、RT-PCR. A total of 106 of the differentially displayed TDFs werecloned and sequenced, and were identified as homologues to the Arabidopsis genesclassified into twelve categories, viz, transcription factors, stress respon

20、sive genes,genes regulating fatty acid metabolism, and genes relating to signal transduction andso on. 34 and 39 of them were GA-and MeJA-responsive, respectively. A total of 24TDFs were both GA3- and MeJA-responsive, su

21、ggesting a cross-talk of the two plantgrowth regulators in modulating the development of oilseed rape flower. Male sterility is interested in the utilization of heterosis that means an efficientmethod to promote oilseed

22、 rape production. Various kind of heterosisutilizationsystems, including the cytoplasmic male sterility (CMS) system (such as pol-CMS,Shan2A-CMS, ogu-CMS, tour-CMS, nap-CMS), the nuclear gene male sterility(GMS) system,

23、the self incompatibility (SI) and the chemical induced male sterility(CIMS), have been established. Each system has its advantageous and its clearinapplicability. In Chapter 3.4 and 3.5, we made valuable efforts to estab

24、lish a newsystem of heterosis utilization in oilseed rape, which is based on the discovery of themale sterilities that are maintainable by the application of exogenous GA or MeJA.Such male sterilities were sought through

25、 both genetic engineering approach(Chapter 3.4) and traditional EMS mutagenesis (Chapter 3.5). BnDAD1, a putativeBrassica napes orthologue to Arabidopsis DAD1, was isolated and cloned. Molecularcassettes containing the p

26、romoter of BnDAD1 and antisense orientation of theBnDAD1 coding region was constructed in a binary vector pCAMBIA1300, which isused to transform Agrobacteria and rapeseed cultivar `Zhe-You 758'. On the otherhand, EMS mu

27、tagenesis resulted in a total of 75 M1 individual plants that could bemaintained by the exogenous stimulations like thermal water, and MeJA or GA3. Themale sterility of the M1 lines was maintained by crossing with fertil

28、e plants. The malesterile descends were further characterized in the M3 generation for their anther andpollen development under microscope and the ability to set seeds under theapplications of various concentrations of M

29、eJA or GA3 growth regulators. Taken together, we described the flowering time, floral organ morphology thatwere affected by exogeneous MeJA and GA3 in oilseed rape, and characterized themolecular mechanism underlying th

30、e phenotypic appearance. Our results could be anenriching addition to the body of work that attempts to understand the signalingfunction of MeJA and GA in the floral inductive pathway in oilseed rape. The start-upwork to