Tomato root knot nematode disease is one of the most important diseases in tomato production. It will cause huge economic losses of [1]. Root knot nematodes infecting tomato, mouth needle feeding on plants caused by mechanical damage; at the same time, nematode secretions and excretions will affect plant metabolism, the tomato cell expansion, nuclear division, forming multinucleated giant cells, and the formation of local tumor like root node in the plant roots and plants for water and nutrient absorption; in addition, root knot nematode feeding and in the root activity caused by the wound will become other soil spread pathogens invading channel and promote the occurrence of other diseases [2].
By physical control, chemical control, biological control and other methods can reduce the damage of root knot nematode to a certain extent, but the effect is not good, and even can cause damage to the environment, the most effective method of production is the cultivation of tomato varieties resistant to root knot nematode [3-4]. There are 9 genes that have been identified in tomato resistance to root knot nematode, respectively, Mi-1, Mi-2...... Mi-9, only the Mi-1 gene contained in tomato varieties, the gene was introduced into Tomato by embryo rescue, while the other 8 genes remained in the wild tomato [5-7]. Mi-1 plays a great role in the 60 years of the cultivation of tomato, but the gene at the soil temperature is higher than 28, the loss of resistance to the root knot nematode, which has a huge impact on the summer tomato cultivation and protection in China [8]. In wild tomato, some resistance to root knot nematode gene has the heat stable. The Mi-3 and Mi-9, and Mi-1, Mi-9 and [9-10], are the same. However, the wild tomato with the heat stable disease resistance gene was not compatible with the cultivation of tomato. The gene was not successful. Although the method of embryo rescue was successful, the fertility of the plants was affected, and the gene was not applied to tomato varieties [11-12].
Through the introduction of tomato materials and resistance to disease resistance, a tomato material was collected from Yunnan, which has been shown to have a good resistance to the root knot nematode. The material is ZN17, which has important significance for alleviating the problems encountered in the production of [13]. By genetic analysis and gene mapping of F2 populations constructed by ZN17 and 09C84, the single gene in ZN17 was confirmed to be a characteristic of heat stable and resistant to root knot nematode, which is located in the short arm of chromosome 6.
At present, there is no report on the cause of the loss of resistance to Mi-1 at high temperature. The expression of Mi-1 was detected by using the specific primers of Mi-1 gene, and the cause of the loss of resistance at high temperature in Motelle was elucidated. In addition, Mi-9 and Mi-9 were used to study the relationship between Mi-HT and Mi-1, and the Mi-HT silencing and Mi-1 were used to analyze the relationship between Mi-1 and gene, and to further study the relationship between Mi-1 and Mi-9, and to explore the relationship between gene silencing.
1 materials and methods
1.1 root knot nematode
The root knot nematode used in the experiment is the root knot nematode in the south, and the research group of the Department of plant diseases, China Agricultural University, is presented. Egg masses will root attached to the pick into the improved Berman funnel, at 28 DEG C incubator for root knot nematode second instar larvae hatching. When the concentration of inoculation fluid reached the inoculation concentration, the plant was used for the inoculation, and the two 3000 instar larvae were inoculated with the [15].
1.2 plant materials
4 tomato accessions were: root knot nematode wild tomato materials LA2157 resistance of heat stable resistance to root knot nematode tomato materials ZN17 Mi-HT/Mi-HT, thermal stability Mi-9/Mi-9, thermosensitive inductive reactance of the root node nematode tomato materials Motelle Mi-1/Mi-1 and susceptible to root knot nematode tomato materials 09C84 mi-1/mi-1.
1.3 resistance identification
After inoculation with root knot nematode 40 days, on the high temperature of 32 degrees C and at room temperature 23 to 26 DEG C under the condition of the tomato material of egg mass counts, using SAS data difference significant analysis.
1.4 expression analysis sample preparation
When two leaves were transplanted into 10 cm (diameter) x 10 cm (height) of the nutrition bowl, a part of the seedlings were placed in the culture box for high temperature expression. After transplanting 10 d, the temperature of the incubator was slowly increased, and the temperature reached 32 after 7 d. The seedlings of 2 at 32 D were inoculated with the same volume of distilled water at high temperature and high temperature, and the root knot nematodes were inoculated with the same volume of distilled water. Sampling plant gently removed, with distilled water for cleaning root soil and impurity, with a pair of scissors cleaning clean the root apical tip 2 cm cut, wrapped in aluminum foil, label sample name, temperature, time, after quickly into liquid nitrogen frozen, and then put into the 80 DEG C ultra low temperature freezer for sample storage. 3 D, 9 d were sampled, and 2 strains were sampled at each temperature.
1.5 RNA extraction and RT-PCR analysis
RNA was extracted with Promega kit, and the RNA was prepared by using TaKaRa reverse transcription kit into cDNA. Using cDNA and DNA as the template, the PDS gene specific primers were used to detect the DNA contamination. In determining the DNA pollution, with the Ubi3 gene of the cDNA standard homogeneous concentration.
RT-PCR amplification primer is: positive to /2Do-F C1, to C2S4-R[16], the primer can be used to amplify the Mi-1 gene. The reaction system is as follows: DNA 2 L, 10 * buffer PCR 2 L, dNTPs (10 mmol / L) 0.4 L, DNA Polymerase L (5 U / rTaq) 0.2 L, l 0.2 ddH2O. The PCR amplification program was as follows: 4 94 min, 45 s, 45 s, 45, 72 s, 23, 6 min, 72, 4.
Construction of recombinant vector mediated gene silencing mediated by 1.6 virus
Using primers C1/2DO-F/ C2S4-R and PDS-F/R cut at the 5 'end ligase sites and protective bases (Table 2). Motelle cDNA as the template, on tomato Mi-1 and PDS gene fragment was amplified. The amplified product was connected to pMD18-T, and the positive clone plasmid was extracted. VIGS vector used in this study was constructed by rattle virus Tobacco (TRV). Using XbaI and BamHI double enzyme digest the cloned plasmids and TRV2 and recovery were small fragments and fragment and connect the plasmid with T4 DNA ligase. In this study, the use of TRV vector for Professor Liu Yule.
2 knots
2.1 the incidence of Tomato in different temperature conditions
40 d after inoculation, the high temperature and normal temperature under different materials after the onset of egg growth status and statistical significance analysis. The results (Table 3) showed that, under normal conditions, 09C84 digital egg than ZN17, Motelle and LA2157. Under the condition of high temperature, the number of egg masses Motelle increased, significantly lower than the susceptible materials, and significantly higher than that of ZN17 and LA2157.
Sequence analysis of gene expression fragments of 2.2 tomato materials
CDNA and genomic DNA (positive control) were amplified by PCR using PDS primers, and the sequence of 6kp was 1 min, and the sequence of PCR was 2. Taking the 09C84 test result as an example, it can be used to determine the cDNA of the DNA, which is not contaminated by genomic DNA.
The products of Motelle, ZN17 and LA2157 were sequenced and analyzed. In order to determine the accuracy of the sequencing and the detection of other homologous sequences, 3 different clones were sequenced, and the sequence was determined by comparing the 3 sequencing results. The sequencing results showed that the sequence information of the 3 clones was exactly the same as that of the different gene fragments. Motelle and ZN17 amplified fragment sequences were exactly the same, and the sequence alignment showed that the sequence of the amplified sequence was 95.95% similar to that of LA2157.
RT-PCR analysis of tomato materials under 2.3 different temperatures
The expression of cDNA was firstly carried out, and then the expression of root knot nematode was analyzed after different temperature. The expression of Mi-1 gene was detected by Motelle at normal temperature and 3 D and 9 D, but no significant difference was detected between inoculated water and inoculated root knot nematode, and the expression of Mi-1 gene was not detected in high temperature conditions. The expression of Mi-1 gene was detected by LA2157 and ZN17, and there was no significant difference in the expression of gene in 3 D and 9, and the expression of Mi-1 was similar to that of D. The expression of ZN17 and LA2157 in the high temperature was lower than that in normal temperature. The expression of the related genes was detected at high temperature and room temperature, and the 09C84 was not listed in Figure 3.
2.4 VIGS recombinant plasmid vector construction
By expression analysis, Mi-1 specific primers can be amplified to homologous gene fragments in each material, and the expression changes of genes are consistent with the resistance of different materials. In order to further explain the association between the genes, this study was used to construct the VIGS recombinant vector for future research. The viral vector used in tobacco is the tobacco rattle virus (TRV), which belongs to the RNA virus, which is composed of RNA1 and RNA2 two chains. After the transformation of the viral vector including pTRV1 and pTRV2 two independent plasmid structure, respectively, loading RNA1 and RNA2 genetic information. Objective to construct the multiple cloning site of pTRV2 in pTRV1, which is responsible for the transport of virus in plant tissues. In order to verify whether the obtained plaque is a recombinant plasmid containing the plaque, the primers were designed to identify and sequence the pTRV2 and its recombinant vector. According to the sequence of RNA1, primers were designed to verify the pTRV1 vector. In addition, the recombinant vector was confirmed by sequencing and sequencing analysis.
3 discussion
Through the identification of disease resistance and statistical analysis of Tomato in different materials, under the condition of normal temperature and Motelle on the root knot nematodes has good resistance, but when the soil temperature reached 32 degrees, egg masses display a increased, while the number of egg masses is still less than the susceptible control material 09C84, but the combination of the appearance of the plant growth, root node number, the number of egg masses were analyzed. The material by the room temperature resistance traits change for high temperature susceptible traits, lost to root knot nematode disease resistance were determined. ZN17 and LA2157 showed high resistance to high temperature and normal temperature. ZN17 is a cultivated tomato material, which can be transferred to tomato varieties by conventional breeding methods to solve the problem of the loss of resistance to Mi-1 in high temperature.
The expression of Mi-1 gene at different temperatures was analyzed, and the expression of Mi-1 gene could be detected when the soil temperature was less than 28, and the expression of cDNA gene was not detected in the reaction system. At the same time, the gene expression product was not detected at high temperature and normal temperature, and the Mi-1 and Motelle were consistent with 09C84 and 09C84 in the high temperature and normal temperature. Because of the presence of Mi-1 gene, Mi-HT and Mi-9 were also located in the region of [14], and the expression of genes related to these 2 genes were also analyzed. By comparison of the amplification sequence, the sequence of the obtained sequence was homologous with Mi-1 gene. The expression of related genes in LA2157 and ZN17 was analyzed, and it was found that the expression of gene was detected in both normal and high temperature, and the resistance to root knot nematode was consistent with the material at different temperatures. If the amplified fragment is Mi-HT and Mi-9, the Mi-1, Mi-9 and Mi-HT are the same. In addition, the expression level of all the detected genes was not changed by the analysis of the expression of the different time, and the inoculation of the nematode, which belongs to the gene [17]. Under the condition of high temperature, the expression of ZN17 and LA2157 genes decreased slightly, and it also coincides with the increase in the number of egg masses under high temperature.
Through the research of the expression and the analysis of gene sequences, the expression of Mi-HT, Mi-9 and Mi-1 in the 3 root knot nematode resistance genes could be deduced. Although ZN17 and Motelle are the same as that of, the Mi-HT gene has not been cloned, and the sequence of the downstream sequence is the same as that of Mi-1, and of course, it does not rule out the possibility of ZN17 in the regulation of Mi-HT expression. Using the constructed recombinant vector pTRV2-Mi, the Mi-HT gene was studied. It can be explained that the gene is homologous to Mi-1, and the work is being carried out. Further study on the mechanism of the heat stable resistance to root knot nematode can not only clarify the regulation mechanism of the gene, but also play a great role in the utilization of resistance genes.