| Phylloxera(Daktulosphaira vitifoliae Fitch) is the obligate parasitic pests on grapevine. In2005, phylloxera was found again in China which made a great threat to the development of grape industry and the winged and leaf-gall aphids will accelerate the spread of phylloxera, so studying phylloxera wing polyphenism is very significant. In this paper, we cloned and analyzed Apyrase gene which is related to phylloxera wing polyphenism and also studied its expression difference in any instar of winged and wingless phylloxera by using the FQ-PCR technology. Meanwhile, we studied the influence of hormones and environmental factors on phylloxera wing polyphenism. The main results were as follows:1. Cloning and analysis of DvApyrase gene related to phylloxera wing polyphenismThis experiment first cloned the related gene of phylloxera wing polyphenism and analyzed it. This gene belongs to Apyrase superfamily and we named it Dv Apyrase whose GenBank accession is KJ725377. The total sequence length of DvApyrase gene cDNA is1199bp, including a1071bp ORF,63bp5’ UTR and65bp3’UTR. The ORF codes a polypeptide about356aa and its molecular weight is39.68kD. The amino acid sequence of DvApyrase has hydrophobic profiles between1to20aa but not exist transmembrane regions, so we conclude DvApyrase may be a kind of membrane protein. The Apyrase homology of phylloxera and other insects is about40%-50%by homology comparison. According to the evolutionary analysis, we find that DvApyrase of phylloxera and SCAN of Acyrthosiphon pisum cluster to one branch in the cladogram evolutionary tree so that their relationship is the nearest.We used the FQ-PCR technology to analyse the relative expression of DvApyrase in any instar of winged and wingless phylloxera, and the results showed that DvApyrase expression quantity of winged phylloxera were significantly higher than the wingless’s and reaching peak at winged age2which is13.62times as much as the wingless, then expression quantity declined with the increase of winged age gradually. So it suggests that DvApyrase gene plays a key role on phylloxera wing polyphenism.2. Analysis on effects of hormones on phylloxera wing polyphenismThe effect of Precoceme Ⅱ to wing polyphenism:The results showed that the wing polyphenism rate was observably higher than the control and the winged rate rised significantly along with the increase of the concentration of the PⅡ under the concentration of lng/(μl-100ng/μl by topical application. Also, the wing polyphenism rate was not observably different from the control when the concentration was lng/μl by feeding method. Only when the concentration increased to100ng/μl, the wing rate increased significantly. Thus, we conclude that Precoceme Ⅱ can promote phylloxera wing polyphenism.The effect of JHⅢ to wing polyphenism:The results showed that the wing polyphenism rate was observably lower than the control under the concentration of O.lng/ul and lng/ul by topical application, and the wing rate also continued to decrease along with the increase of concentration but not significantly. But, the wing polyphenism rate was observably declined when the concentration increased10ng/μl by feeding method. All in all, Juvenile Hormone Ⅲ has inhibitory effects on phylloxera wing polyphenism.3. Analysis on effects of environmental factors on phylloxera wing polyphenismIn order to research the effects of environmental factors on wing polyphenism of phylloxera, the effects of density, temperature and host on wing polyphenism were studied by using1instar nymph of phylloxera. The results showed that the wing polyphenism rate of phylloxera increased with the raise of nymph density. The wing polyphenism rate also raised with the temperature rising within a certain range and peaked at28℃(31.24%), but it declined at32¤significantly. The wing polyphenism rates existed disparity on different varieties. The wing rate of Cabernet Sauvignon and Kyoho was28.40%and26.40%, and Vitis davidii Foex and Beta was23.00%and21.76%. |
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