| In order to demonstrate the existence of 5-hydroxytryptamine(5-HT)in cotton,and explore the spa-tial and temporal differences of 5-HT expression in different varieties(lines)of cotton and its relationship with insect resistance of cotton.In this experiment,a HPLC method for the determination of 5-HT in cotton was first established by referring to other detection methods in plants and animals.Using this method,5-HT content was determined in cotton samples of different varieties(lines)and different periods planted in the field,and samples of different varieties(lines),different periods and before and after Be-misia tabaci(Gennadius)feeding in the room.And the relationship between the content of 5-HT and the insect resistance of cotton was studied by bioassay of Helicoverpa armigera(Hübner)by feeding with exogenous 5-HT.Then,the candidate genes involved in 5-HT synthesis in Gossipium hirsutum were screened based on the reported CYP genes involved in 5-HT synthesis,and the gene function was identi-fied by yeast recombinant expression.The main results are as follows:1.A HPLC method for the determination of 5-HT in cotton leaves was established.The detection results of 5-HT showed good linearity in the range of 0.5μg/m L～100μg/m L,with R~2 value of 0.9999;the detection limit was 0.1μg/m L,and the quantitation limit was 0.5μg/m L.The relative standard devi-ations(RSD)of intraday and inter day precision tests were less than 3%,and the recoveries of samples ranged from 95%to 102%.The relative standard deviations of samples with different concentrations were less than 3%,and the average relative standard deviation was 1.56%.The results showed that this HPLC method had high accuracy,high sensitivity and low interference,which was suitable for the determination of 5-HT in cotton samples.2.The content of 5-HT in different cotton varieties(lines)at different growth stages was detected by HPLC.The results showed that there were significant differences(P<0.05)in the content of 5-HT among the strains related to the resistance of Apolygus lucorum(Hemiptera:Miridae).The content of 5-HT showed that aomian 618>08 shengshi 5>Jimian 616>yazhoumian.However,there was no difference among the varieties with different resistance to B.tabaci.The temporal and spatial dynamic expression analysis of 5-HT in different varieties showed that both B.tabaci resistant strains and A.lucorum resistant strains showed the highest 5-HT content in leaves at budding stage,then decreased gradually at flowering stage,and decreased like seedling stage at bolling stage.The results showed that the expression of 5-HT in cotton was negatively correlated with its insect resistance,and the content of 5-HT also changed with the development of cotton plant,with the highest significance in budding stage.3.The content of 5-HT in cotton leaves before and after feeding by B.tabaci was detected by HPLC.The content of 5-HT in cotton with three different resistances to B.tabaci was 4.46μg/g、10.30μg/g and8.88μg/g,however,which rise to 20.77μg/g、17.54μg/g and 10.69μg/g after feeding by B.tabaci with24 h.There were significant differences in serotonin content between Kelin 13-68 and Kelin 13-93 before and after feeding by B.tabaci.The difference about Kelin 13-68 was very significant(P<0.01),and the content of 5-HT increased by 4.65 times and Kelin 13-93 increased by 1.70 times(P<0.05).The results showed that the feeding of B.tabaci significantly increased the content of 5-HT in cotton,and the sensitive strain of B.tabaci was more sensitive.4.Bioassay of serotonin on H.armigera by mixed feed method.The results showed that exogenous serotonin could shorten the pupation cycle of H.armigera.The peak of pupation in the control group was about 17 days after egg incubation.After treatment with 5-HT,the peak of pupation was advanced to 15days after hatching.In addition,the addition of 5-HT could significantly increase the average pupal weight of H.armigera(P<0.01).In general,the extra intake of 5-HT is beneficial to shorten the generation cycle of H.armigera,enhance physiological performance and harm to cotton.5.Based on the reported CYP71A1 genes involved in the synthesis of 5-HT in rice,the candidate genes involved in the synthesis of 5-HT in Gossypium hirsutum were screened,and the genes with high similarity are Cot AD＿50968、Cot AD＿22317、Cot AD＿66315 and Cot AD＿01894.The gene function of them was identified by yeast expression in vitro.It was found that the five selected genes did not express T5H tryptamine-5-hydroxylase and could not catalyze the production of 5-hydroxytryptamine. |
PDF Full Text Request