Molecular Cloning and Analysis of Expansin Gene from Heterodera Glycines and Evaluation of Soybean Germpalsms Resistance
|School||Nanjing Agricultural College|
|Keywords||Heterodera glycines expansin Southern blot In situ hybridization race resistance evaluation|
Soybean cyst nematode (SCN), Heterodera glycines, is an obligate endoparasitic nematode in plant roots. The second stage juveniles invade the roots of soybean, induce the metabolism imbalance and injure the root tissues, which lead to plant diseases and yield losses. H. glycines was first discovered in 1899 in China and happened in the major soybean production countries of the world such as American, China, Brazil and Japan. Soybean cyst nematode is also called Huolongyangzi in Chinese. SCN causes the most serious damage to soybean production around the world.In previous research, expansin was considered as a kind of protein seperated from plant in vitro by inducement and could expand the plant cell wall. It only existed in plants and could control the expansion of plant cell wall during the growth of the plant cell. Furthermore, expansin could also modulate the upgrowth of the plants and was a component that determined the development in the cell wall.A kind of protein which was high homologous with expansin of plants was discovered by Qin et al. (2004) when they identified the secreted proteins from the infective second stage juveniles of Globodera rostochiensis. This nematode originated protein could induce the plant cell wall to expand in the experiment in vitro. Many recently studies demonstrated that the expansin genes are probably existed in many plant parasitic nematodes. No expansin genes have been reported in H. glycines. Therefore, the two new expansin genes from H. glycines were cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) and their sequences were analysed. The copy number in genome was detemined by southern blot and the expression site of gene in nematode body was demonstrated by in situ hybridization. The research provided a basis for better understanding the pathogenic mechanism of expansin and a clue for the strategy for controlling H. glycines.According to the differences in reproductive capacity on differential hosts, H. glycines can be separated into 16 different races by their virulences. A lot of efforts had been utilized to control H. glycines, such as physical or chemical methods, biological control, or agricultural measures etc., among which the breeding of resistant cultivars was the most economic and effective method. The screening resistance of resistant soybean varieties and germplasms to H. glycines is the basis for breeding new resistant cultivars, therefore, the resistance of soybean varieties and germplasms to Race 3 from Heilongjiang province and Race 4 from Langfang of Hebei province were evaluated in the study. The results provided the basic knowledge of the resistance level of soybean germplasm resources in our country and evidence for the breeding and utilization of resistance varieties.1. Molecular cloning and characterization of new expansin genes from H. glycinesTwo new expansin genes, Hg-expbl-1 (Genbank assession No. HM798586) and Hg-expb1-2 (Genbank No. HQ853293) were cloned from second stage juveniles of H. glycines. The full cDNA of Hg-expbl-1 is 1 047 bp with an open reading frame (ORF) of 867bp which encodes a deduced protein with 288 amino acids. The length of genomic DNA of Hg-expb1-1 is 2 108bp, contains six introns. The cDNA of Hg-expbl-2 is 1037 long, contains an ORF with 888 bp encoding a deduced protein with 295 amino acids. The genomic DNA sequences of Hg-expbl-2 is 2 927bp, contains six introns too. Southern blot analysis showed that Hg-expbl-1 is probably the multicopy gene or members of a multigene family. In situ hybridization analysis suggested that the transcripts of them accumulated exclusively in the subventral oesophageal gland cells of H. glycines.2. Evaluation of soybean germplasms resistance to race 3 and race 4 of H. glycinesIn order to obtain resistant materials, the evaluation tests were carried out for 300 soybean varieties and germplasms against the race 3 and race 4 of SCN by PVC column method in greenhouse during September 2009 to June 2010. The result from the test to SCN race 3 showed that 12 out of 300 soybean varieties and germplasms were immune,28 were high resistance and 39 were medium resistance, covered the percentage of 4.0,9.3 and 13.0%, respectively. In test to SCN race 4,8 out of 300 soybean varieties and germplasms were immune,18 were high resistance and 51 were medium resistance, with the percentage of 2.7,6.0 and 17.0%, respectively. The comparison of virulence between SCN race 3 and race 4 revealed the obvious differences in 20 tested soybean varieties and germplasms and the virulence of race 4 was stronger than race 3. The identified resistant materials from this study can provide useful information for agricultural production and resistance resource for genetic breeding.