Identification,Detection And Cloning Of ?-1,4-endoglucanase Genes Of Cyst-forming Nematode From Phyllostachys Praecox

Cyst-forming nematode is one of the most important group of plant parasitic nematodes,in which contains many species causing serious losses of agricultural production.During 2014?2016,a systematic study of cyst-forming nematode population from bamboo(Phyllostachys praecox)were made,including morphologcial and molecular identification,molecular species-specific detection,hatching characters,etc.In addition,the full length of cDNA sequence of ?-1,4-endoglucanase gene Hk-eng2 was obtained by RT-PCR and RACE synthesized from the cyst menatode of P.praecox.The main results were as follows:1.Heterodera koreana,a new record of cyst-forming nematode in ZhejiangA cyst-forming nematode population in rhizosphere of P.praecox from Linan,Zhejiang,China was identified as H.koreana based on morphological and molecular methods.All the morphological characters and morphometric data sequences of ITS and D2D3 regeion of 28S RNA gene near identical to that of H.koreana.This is a new record of cyst-forming nematode in Zhejiang province,Eastern China.2.Establishment of a species-specific molecular detection method for H.koreanaThe specific primers Hk/F,Hk/R were designed according to the comparisons of ITS sequences from 23 populations of 10 species of cyst nematode.The duplex PCR method of species-specific molecular detection consists of pair D2A,D3B and Hk/F,Hk/R.PCR products for H.koreana amplified two fragements,with the sequence length of 786 bp and 449 bp,respectively,and the other nematodes only amplified one fragment with the sequence length of 786 bp.The sensitivity of the dectetion method for a single second stage juvenile(J2)was 1/256,and for single cyst was 1/8192.3.Hatching characteristics and viability of J2 of H.koreanaHatching tests were made based on the effect of temperature and presence of Zn2+?Cu2+ and root soil exudates of host.It was found that the proper hatching temperature range are between 20?30?,in which the optimum hatching temperature is 28?.Meanwhile,nearly no hatching at 35?.Zn2+ and root exudates of host stimulates the hatching of eggs with a total hatching amount of about 1.2 times of the control,while Cu2+ inhibit the hatching of eggs with total hatching amount of about 0.5 times of the control.Effect of temparature on viability of J2 of H.koreana were tested under 6 different temparatures,including 5?,20?,25?,28?,30? and 35?.It was found that the viability of J2 were decreased with the increase of the tempearature.Low temperature is proper for survival of J2.4.Cloning of full length cDNA sequence of ?-1,4-endoglucanase genes of H.koreanaThe full length cDNA sequence of ?-1,4-endoglucanase genes was obtained by RT-PCR and RACE from H.koreana,named as Hk-eng2(GenBank:KY314987).It consists of 1658 nucleotides,29 bp 5'-UTR and 138 bp 3'-UTR,with a 1488 bp ORF,encoded 496 amino acids.The putative protein with PI 7.15 and the oretical molecular weight 51.7 kDa was classified as a member of glycosyl hydrolase family 5.Sequence analysis showed that there are 15 amino acid signal sequence at N terminal and a bacterial type cellulose-binding domain? at C terminal(CBD ?).