Identification of guava root-knot nematodes in Guangzhou and the cloning of 14-3-3 gene

FENG Yan; RUAN Xiancong; CHEN Jun; LUO Mei; LIU Shuxian; JU Haiyan; XIE Zhonghui; XU Shijin

doi:10.13925/j.cnki.gsxb.20170148

Abstract：[Objective]To make sure what species the root knot nematode was that affected the guava in Guangzhou,six guava cultivation areas affected by root knot nematodes were investigated.In addition,to study the pathogenic factor,the 14-3-3 gene was cloned from the identified Meloidogyne enterolobii.[Methods] The females were separated from the diseased roots of guava,and the second instar larvae were incubated from the eggs separated from the roots.The perineal were cut manually under an anatomical microscope,and then the perineal patterns were observed under microscope.The males were separated from the soils of diseased guava using the Baermann method.Then,the female,male and the second instar larvae were observed under optical microscope using the morphological method to identify the root knot nematode species they belonged to.Molecular biology method was also applied for further verification of the molecular results.The primer set #C2F3 and #1108 were used for mtDNA PCR amplification.Then,the degenerate primers were designed according to the 14-3-3 gene of M.incongnita and M.hapla.RT-PCR was performed to amplify the 14-3-3 gene using the designed primers.The PCR product was run with electrophoresis.The fragment was cloned and sequenced.The sequence was analysis by bioinformatics method.Sequence analysis of genes and proteins was carried out using Blast for sequence similarity analysis at NCBI.Open reading frame (ORF) was analyzed with ORF Finder ( physicochemical properties were predicted by EXPASY ( analysis using TMPRED forecast ( form.html).Protein translation and motif analysis were performed using proteomic on-line ExPASy.[Results] The morphological and molecular characteristics of guava root-knot nematodes from diseased root tissue of guava in Xintianer village and Lianan village of Huadu district,Guangzhou and Shixia village of Zengcheng district,Guangzhou,were monitored.The root knot nematodes including female,male and the second instar larvae were observed under optical microscope.The second instar larvae were worm shaped,body ring was small and clear.Side lips were large triangular with lip slightly higher than the middle lip.The mouth needle was slender,conical whole slender,blunt head,crown obvious,sharp tail.The slender needles of the two instar larvae were able to be clearly seen.The base of the stylet was clear,large and round,the middle esophagus line and middle esophagus ball are clear,and the middle esophagus ball was oval.The female body was pear shaped with a prominent neck.Lips was discoid,with slightly rose.The mouth needle was slender,with a large baseball.The perineum was ovoid or oval in shape,the lines were fine and smooth,the tip of the tail was in irregular ring shape,and the lateral line was not obvious.The male was worm shaped,with a clear head ring.The head was high round,slightly constricted,apex cone.The middle esophagus ball was oval.The tail was short and round.Transition was curve.The morphological characteristics showed that they were M.enterolobii.With molecular biological techniques,primer #C2F3 and #1108 were used for mtDNA PCR amplification.The amplification product was near 750 bp.Both the morphological and molecular results showed that the guava root-knot nematode was M.enterolobii.Degenerate primers were designed according to the 14-3-3 gene of M.incongnita and M.hapla.The results showed that the open reading frame of the M.enterolobii 14-3-3 gene contained 783 bp fragments,encoding a protein of 261 amino acids.It was named as Me-14-3-3.The resulting sequence was compared to blastx on NCBI,and the results showed that the 14-3-3 gene was the same as expected.The top two genes with the highest similarity were the 14-3-3 genes from M.incognita (accession number:AAL40719) and Aphelenchoides besseyi (accession number:AJE60959),with the similarity of 98％ and 80％,respectively.The molecular mass of 14-3-3 protein was predicted with Protparam tools to be 29.6 ku.The theoretical isoelectric point was 4.67,and the molecular formula was C1296H2065N337O424S13,with a total of 4 135 atoms.The stability coefficient was 46.60,suggesting that the protein was not stable.It had the unique domain of 14-3-3 gene through analysis.The 14-3-3 sequences of M.incognita (AALA0719 and AAR85527) were downloaded.Two M.hapla 14-3-3 sequences (2475 and 1597) were obtained by comparison with the transcriptome database of northern root knot nematodes.The 14-3-3gene cloned from M.enterolobii was the same group as the AAL40719 from M.incognita and the 1 597 gene from M.hapla.By comparison,most of the three 14-3-3 genes with the same group were conserved,but there were still 8.92％ variations.[Conclusion] All the root-knot nematode that separated from the diseased guava in Guangzhou were M.enterolobii.The M.enterolobii 14-3-3 gene was successfully cloned,and the M.enterolobii sequence was analyzed with some tools on internet.