CN116445540A - Construction method of sweet potato leaf curl virus infectious clone and efficient and simple sweet potato infection method - Google Patents
Construction method of sweet potato leaf curl virus infectious clone and efficient and simple sweet potato infection method Download PDFInfo
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Abstract
The invention discloses a construction method of sweet potato leaf curl virus infectious clone and a high-efficiency simple infection method of sweet potato. 1.01 repeated sweet potato leaf curl virus genome sequences are inserted into a binary vector to obtain sweet potato leaf curl virus infectious clone. The invention provides a method for solving the problem of low infection rate of sweet potato by inoculating sweet potato leaf curl virus into a natural host. Through observation of disease symptoms and detection of PCR, the invention successfully improves the inoculation rate of the sweet potato leaf curl virus to different varieties to 100 percent. In addition, the method provided by the invention is not only efficient, but also easy to operate, can realize large-scale inoculation and screening, and can provide powerful support for subsequent screening of resistant varieties and research on interaction of sweet potato leaf curl virus and sweet potato.
Description
Technical Field
The invention relates to the fields of genetic engineering and plant virology, in particular to a construction method of sweet potato leaf curl virus infectious clone and a high-efficiency simple infection method of sweet potato.
Background
The sweet potato has the functions of grain and cash crops, has wide application and very important economic value. China is the largest sweet potato producing country in the world. In recent years, diseases and insect pests of sweet potatoes, particularly viral diseases, are increasingly occurring. The sweet potato belongs to asexual propagation crops, and the virus is easy to infect in the generation propagation. Among them, sweet potato leaf curl virus (Sweet potato leaf curl viru s, SPLCV) is the most predominant sweet potato DNA virus. To study the interaction between virus and host, the primary condition is to obtain efficient, stable, reproducible vaccination methods under laboratory conditions. Traditional grafting inoculation methods are difficult to control and infect single viruses, and symptoms are easily affected by various factors, so that the stability and the repeatability are poor. Sweet Potato Leaf Curl Virus (SPLCV) is a single-stranded DNA virus belonging to the family Geminiviridae (Geminiviridae), the genus Begomovirus. At present, most geminivirus infectious clones are obtained by connecting 1.2-2.0 virus genomes in tandem to an agrobacterium binary vector, and then performing virus inoculation by using an agrobacterium-mediated leaf penetration or stem injection method. Nevertheless, the research on the interaction relationship between SPLCV and sweet potato is still basically in a blank stage, and the main reason is that the construction of the SPLCV invasive clone is imperfect, the inoculation method is not mature, and the highest efficiency of the reported inoculation method can only reach about 20%.
Disclosure of Invention
The invention aims to provide a construction method of sweet potato leaf curl virus infectious clone and a high-efficiency simple infection method of sweet potato.
The construction method of the sweet potato leaf curl virus infectious clone comprises the step of inserting 1.01 repeated Sweet Potato Leaf Curl Virus (SPLCV) genome sequences into a binary vector to obtain the sweet potato leaf curl virus infectious clone.
Preferably, 1.01 repeated Sweet Potato Leaf Curl Virus (SPLCV) genome sequences are inserted into the multiple cloning site of a binary vector pCAMBIA1300 transformed by agrobacterium to prepare pCAMBIA1300-SPLCV-1.01 invasive clone.
Preferably, the 1.01 repeated sweet potato leaf curl virus is a stem loop structure of a key sequence related to virus replication repeated at the tail end of the genome sequence of the sweet potato leaf curl virus.
Preferably, a key sequence stem loop structure related to virus replication is repeated at the tail end of a sweet potato leaf curl virus Guangzhou strain virus (SPLCV-GZ 02) gene and is connected to an agrobacterium-transformed binary vector pCAMBIA1300 through KpnI and XbaI double enzyme cutting sites to obtain pCAMBIA1300-SPLCV-1.01 invasive clone, wherein GenBank accession number of the sweet potato leaf curl virus Guangzhou strain virus is JX286654.
Preferably, the nucleotide sequence of the stem-loop structure is AAGGCGGGCACCGTATTAATATTACCGGTG CCCGCCGCGCC, which is 41bp.
The invention also provides a method for efficiently and simply infecting sweet potatoes by utilizing the sweet potato leaf curl virus infectious clone, which is to introduce the sweet potato leaf curl virus infectious clone into an agrobacterium AGL1 strain to obtain recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone; soaking sweet potato seedlings in 6-8 leaf period in recombinant agrobacterium liquid containing sweet potato leaf curl virus infectious clone, transferring to clear water for continuous soaking, and culturing in transplanting matrix.
The sweet potato leaf curl virus infectious clone is pCAMBIA1300-SPLCV-1.01 infectious clone.
Preferably, the soaking of the sweet potato seedling in the 6-8 leaf period in the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone is to soak the sweet potato seedling in the 6-8 leaf period and the length of 15cm in the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone for 6 hours, and then transfer the sweet potato seedling to clear water for continuous soaking for 12 hours.
Preferably, the sweet potato seedling is cultured in a transplanting matrix, the redundant leaves are cut off, only 2-3 leaves are reserved, and the sweet potato seedling is transplanted into a matrix mixed with nutrient soil, perlite and sand in a volume ratio of 3:1:1 for continuous culture.
Preferably, the sweet potato seedling in the 6-8 leaf period is soaked in recombinant agrobacterium tumefaciens bacteria solution containing sweet potato leaf curl virus infectious clone, and the vine of the sweet potato seedling is cut into a fresh section with the angle of 45 degrees by a knife before the sweet potato seedling is soaked.
Preferably, the concentration of the recombinant agrobacterium solution containing sweet potato leaf curl virus infectious clone is OD 600 ＝1.0。
Preferably, the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone is prepared by culturing recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone in LB culture medium with corresponding resistance overnight, re-suspending the agrobacterium in complete induction culture medium, and re-suspending the agrobacterium after overnight culture 1 / 4 And (3) soaking in a buffer solution for soaking and inoculating sweet potato seedlings.
The invention is characterized in that the stem-loop structure sequence in the genome of sweet potato leaf curl virus (sweet potato leaf curl virus, SPLCV) is repeated once at the tail end, and is constructed into pCAMBIA1300 vector, thus obtaining the minimum unit and efficient invasive clone of sweet potato leaf curl virus. The infectious clone can successfully infect different varieties of sweet potatoes by an agrobacterium tumefaciens AGL1 mediated efficient and simple agrobacterium tumefaciens soaking inoculation method. The invention provides a method for solving the problem of low infection rate of sweet potato by inoculating sweet potato leaf curl virus into a natural host. Through observation of disease symptoms and detection of PCR, the invention successfully improves the inoculation rate of the sweet potato leaf curl virus to different varieties to 100 percent. Moreover, the method provided by the invention is not only efficient, but also easy to operate, can realize large-scale inoculation and screening, and can provide powerful support for subsequent screening of resistant varieties and research on interaction of sweet potato leaf curl virus and sweet potato.
Drawings
Fig. 1: SPLCV-1.01 sequence diagram. The framed "TATTAATATTA" sequence is a conserved sequence of geminiviruses. Wherein the thickened "A" is the replication origin of the sweet potato leaf curl virus. The underlined 41bp sequence is the repetitive stem-loop structure necessary at both ends of the invasive clone.
Fig. 2: schematic of the invasive cloning of pCAMBIA 1300-SPLCV-1.01.
Fig. 3: disease symptoms of broad-potato 87 inoculated with sweet potato leaf curl virus. The control group was inoculated with Agrobacterium containing the empty vector of pCAMBIA1300 and the experimental group was inoculated with Agrobacterium containing the invasive clone pCAMBIA 1300-SPLCV-1.01.
Fig. 4: PCR detection of viral DNA content after 20 days (20 dpi) of inoculation of 10 Kadsura pepper 87. The viral AV2 gene, ibelF gene, was detected by PCR as an internal control. Water (H) 2 O) and the broad-potato 87 plants (Mock, M) inoculated with the empty vector are used as negative control, and 1-10 are broad-potato 87 plants inoculated with virus.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings.
The specific examples are intended to be illustrative of the invention and are not intended to be limiting. Modifications of the present embodiments, which do not creatively contribute to the invention, can be made by a person skilled in the art after reading the present specification, for example, by enlarging the culture volume correspondingly to accommodate large-scale screening, but are protected by patent laws within the scope of the claims of the present invention.
Examples
A sweet potato leaf curl virus infectious clone and a sweet potato efficient and simple inoculation method comprise the following steps:
1. based on the published genome of the Guangzhou strain of sweet potato leaf curl virus (SPLCV-GZ 02) (GenBank accession number: JX286654, full length 2829 nt), a stem loop (stem loop) structure with a length of 41bp relevant for viral replication is repeated at the end, as shown in FIG. 1 and SEQ ID NO.1, the underlined part in FIG. 1 being the stem loop (stem loop) structure. The full length SPLCV-1.01 sequence of 2870bp is synthesized by gene synthesis technique (FIG. 1). The infectious clone pCAMBIA1300-SPLCV-1.01 of SPLCV is obtained by connecting the KpnI and XbaI double restriction sites to the binary vector pCAMBIA1300 transformed by agrobacterium.
2. The pCAMBIA1300-SPLCV-1.01 infectious clone is quickly frozen by liquid nitrogen, the agrobacterium AGL1 strain is introduced by a heat shock method of heat shock for 5 minutes at 37 ℃, and the recombinant agrobacterium monoclonal clone containing the sweet potato leaf curl virus infectious clone is obtained by screening by 50mg/L kanamycin and 10mg/L rifampicin resistant LB plates and inversely culturing for 2 days at 28 ℃.
3. Recombinant Agrobacterium was selected and subjected to shaking dark culture at 28℃for 10-12 hours in 5mL of LB liquid medium containing 50mg/L kanamycin and 10mg/L rifampicin resistance, to obtain an Agrobacterium culture.
4. 0.5mL of the above Agrobacterium culture was inoculated into 200mL of LB liquid medium containing 50mg/L kanamycin and 10mg/L rifampicin resistance, and shake-dark cultured overnight at 28℃and 200rpm to obtain an Agrobacterium solution.
5. The above overnight-cultured Agrobacterium solution was centrifuged at 3000rpm for 10min, the supernatant was discarded, and the Agrobacterium pellet was resuspended in 500mL of complete induction medium (Table 3) containing 50mg/L kanamycin resistance, and subjected to shaking dark culture at 28℃overnight at 200rpm to obtain an Agrobacterium solution.
6. Centrifuging the above overnight cultured Agrobacterium solution at 3000rpm for 10min, discarding supernatant, and re-suspending the Agrobacterium precipitate in 500mL 1 / 4 MS immersed in buffer (table 4). Detecting OD 600 By using 1 / 4 MS soaking buffer solution to dilute the bacterial solution to OD 600 =1.0, used for sweet potato inoculation.
7. Taking sweet potato seedling with length of 15cm at 6-8 leaf period, cutting vine of sweet potato seedling with knife to obtain 45 degree fresh section, soaking sweet potato seedling in OD 600 In the bacterial liquid of 1.0, after being soaked for 6 hours at room temperature in dark, the sweet potato seedlings are transferred into clear water, and the soaking is continued for 12 hours at room temperature.
8. Cutting off redundant leaves of the sweet potato seedlings, only keeping 2-3 leaves, and then transplanting the sweet potato seedlings into a matrix mixed by nutrient soil, perlite and sand in a volume ratio of 3:1:1 for continuous culture.
9. And respectively taking the new top leaves of the sweet potatoes as materials 10 days and 20 days after inoculation, extracting DNA, and detecting the content of the viral DNA by using PCR.
10. For example, the disease symptoms appear about 20 days after inoculation, using the broad-bean 87. Compared with the control group inoculated with empty vector, the new leaves of the broad-bean 87 inoculated with the virus are yellowing, curled, the growth of terminal buds is obviously inhibited, and lateral buds are increased (figure 3).
11. Taking the broad-bean 87 as an example, DNA of sweet potato leaf curl virus (SPLCV-AV 2) can be detected by PCR in 10 new-born leaves of sweet potato 20 days after virus inoculation (figure 4), and the control group has no virus DNA, which shows that the inoculation rate of the method reaches 100 percent and is far higher than 20 percent of the inoculation rate of the prior reported inoculation method.
TABLE 1
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TABLE 3 Table 3
TABLE 4 Table 4
Claims (10)
1. A construction method of sweet potato leaf curl virus infectious clone is characterized in that 1.01 repeated sweet potato leaf curl virus genome sequences are inserted into a binary vector to obtain sweet potato leaf curl virus infectious clone.
2. The method of claim 1, wherein the 1.01 repeat sweet potato leaf curl virus is a repeat of a viral replication-related key sequence stem loop structure at the end of the genomic sequence of the sweet potato leaf curl virus.
3. The construction method according to claim 1, wherein 1.01 repeated sweet potato leaf curl virus genome sequences are inserted into the multiple cloning site of the binary vector pCAMBIA1300 transformed by agrobacterium to prepare pCAMBIA1300-SPLCV-1.01 invasive clone.
4. The construction method according to claim 1, wherein a key sequence stem loop structure related to virus replication is repeated at the tail end of a virus gene of a Guangzhou strain of sweet potato leaf curl virus, and the key sequence stem loop structure is connected to a binary vector pCAMBIA1300 transformed by agrobacterium through KpnI and XbaI double cleavage sites to obtain pCAMBIA1300-SPLCV-1.01 invasive clone, and GenBank accession number of the virus of the Guangzhou strain of sweet potato leaf curl virus is JX286654.
5. The method of claim 1, 2, 3 or 4, wherein the stem-loop structure has a nucleotide sequence of AAGGCGGGCACCGTATTAATATTACCGGTGCCCGCCGCGCC, which is 41bp.
6. A method for efficiently and simply infecting sweet potatoes by utilizing sweet potato leaf curl virus infectious clones, which comprises the steps of introducing the sweet potato leaf curl virus infectious clone according to any one of claims 1 to 5 into an agrobacterium AGL1 strain to obtain recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone; soaking sweet potato seedlings in 6-8 leaf period in recombinant agrobacterium liquid containing sweet potato leaf curl virus infectious clone, transferring to clear water for continuous soaking, and culturing in transplanting matrix.
7. The method of claim 6, wherein the sweet potato leaf curl virus infectious clone is pCAMBIA1300-SPLCV-1.01 infectious clone.
8. The method according to claim 6, wherein the soaking of the sweet potato seedling with 6-8 leaf period in the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone is carried out by soaking the sweet potato seedling with 6-8 leaf period and length of 15cm in the recombinant agrobacterium solution containing the sweet potato leaf curl virus infectious clone for 6 hours, transferring to clear water and continuously soaking for 12 hours.
9. The method according to claim 6, wherein the cultivation in the transplanting matrix is that the sweet potato seedlings are cut off redundant leaves, only 2-3 leaves are reserved, and the sweet potato seedlings are transplanted into the matrix mixed with nutrient soil, perlite and sand in the volume ratio of 3:1:1 for continuous cultivation.
10. The method according to claim 6, wherein the recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone is prepared by culturing the recombinant agrobacterium containing the sweet potato leaf curl virus infectious clone overnight in LB medium with corresponding resistance, re-suspending the agrobacterium in complete induction medium, and re-suspending the agrobacterium in 1/4MS soaking buffer solution for soaking and inoculating sweet potato seedlings after overnight culture.
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