阅读说明：本技术 C21-孕甾烷及其衍生物在抗植物病毒中的应用 (Application of C21-pregnane and derivatives thereof in resisting plant viruses ) 是由 叶健 郝小江 李玉林 孙艳伟 赵平芝 黄烈军 于 2020-12-15 设计创作，主要内容包括：本发明提供C21-孕甾烷及其衍生物在抗植物病毒中的应用。所述植物病毒包括DNA和RNA病毒,DNA病毒毒具体可为双生病毒科病毒,所述双生病毒科病毒为菜豆金黄色花叶病毒属病毒Begomovirus,亦为粉虱传双生病毒(whitefly-transmitted geminiviruses,WTG),包括单组分病毒(如番茄黄化曲叶病毒)和双组分病毒(如木薯花叶病毒(SiriLa-cassava mosaic virus,SLCMV))。通过接种双组份的双生病毒SLCMV-HN7,发现相对于DMSO对照,GSX3预处理过的植物病症明显减缓,说明GSX3对双生病毒的抗性具有广谱性。(The invention provides application of C21-pregnane and derivatives thereof in resisting plant viruses. The plant virus includes DNA and RNA viruses, and the DNA virus can be particularly geminiviridae virus which is Begomovirus of bean golden mosaic virus, and is also white-transmitted geminivirus (WTG), and comprises single-component virus (such as tomato yellow leaf curl virus) and double-component virus (such as cassava mosaic virus (SLCMV)). By inoculating the double-component geminivirus SLCMV-HN7, the disease of the plant pretreated by the GSX3 is obviously slowed compared with a DMSO control, which indicates that the GSX3 has broad spectrum of resistance to the geminivirus.)

Use of C21-pregnane and its derivatives against plant viruses.

2. Use according to claim 1, characterized in that: the structural formula of the C21-pregnane and derivatives thereof is shown as follows:

3. use according to claim 1 or 2, characterized in that: the plant virus includes DNA virus and RNA virus.

4. Use according to claim 3, characterized in that: the DNA virus is a geminiviridae virus, the geminiviridae virus is Begomovirus of the genus Phaseolus aureus, and is also a whitefly-transmitted geminivirus (WTG).

5. Use according to claim 3, characterized in that: the geminiviridae viruses include monocomponent viruses (e.g., Tomato Yellow Leaf Curl Virus (TYLCV)) and bicomponent viruses (e.g., Cassava Mosaic Virus (CMV)).

6. Use according to claim 3, characterized in that: the RNA virus is rice stripe virus.

7. A method for preventing and controlling plant virus poison comprises the following steps: c21-pregnane and its derivatives are applied to prevent and treat plant virus poisoning.

8. The method of claim 7, wherein: the phytovirus poison is caused by a plant virus;

the plant virus includes DNA virus and RNA virus.

9. The method of claim 8, wherein: the DNA virus is a geminiviridae virus which is Begomovirus of the genus Phaseolus aureus and is also a whitefly-transmitted geminivirus (WTG);

the geminiviridae viruses include monocomponent viruses (e.g., Tomato Yellow Leaf Curl Virus (TYLCV)) and bicomponent viruses (e.g., Cassava Mosaic Virus (CMV)).

10. The method of claim 9, wherein: the RNA virus is Rice Stripe Virus (RSV).

Technical Field

The invention belongs to the field of new pesticide creation, and particularly relates to application of C21-pregnane and derivatives thereof in resisting plant viruses.

Background

Plant diseases and insect pests are main factors for limiting the high and stable yield of crops in agricultural production, and plant virus diseases are called as 'crop cancers'. Due to the lack of effective germplasm resources and control methods against viral diseases, crop viral diseases cause economic losses of as much as 1000 billion dollars per year on crop production. Plant viruses are in a large variety, about 1480 plant viruses are currently reported worldwide, and viruses transmitted by vector insects account for about one third of plant viruses. With global warming and increasingly frequent international agricultural product trade, geminivirus and its transmission vector bemisia tabaci are transformed from small diseases which occur locally in the last 20 years into one of the most important plant virus diseases which are global at present, the caused damage is in a trend of increasing year by year, and is destructive on various crops all over the world, which poses a serious threat to agricultural production.

The bean golden mosaic virus (Begomovirus) is a geminiviridae virus with the most varieties and the most serious harm to crops. They can be classified into monocomponent viruses and two-component viruses according to their DNA genome structures. The two-component virus contains two DNA molecules, DNA A and DNA B such as Cassava Mosaic Virus (CMV). Monocomponent viruses such as Tomato Yellow Leaf Curl Virus (TYLCV), whose genomic structure is only DNA A. The bean golden mosaic virus Begomovirus can only spread by transmission of Bemisia tabaci (Gennadius) in a persistent, recyclable manner, and is also known as whitefly-transmitted geminivirus (WTG). WTGs not only cause direct damage, but also promote damage caused by the occurrence and outbreak of vector insects. In the 70 s of the 20 th century, the host range of geminivirus was narrow and the virus occurrence range was small, so that the geminivirus did not attract much attention of scientists. In the long-term co-evolution of bemisia tabaci and geminivirus, the virus can promote its own spread by regulating the characteristics of bemisia tabaci and the host plant. Researches on Zhouyiping, Liu Tree growth and the like find that the reproductive capacity of the B-type bemisia tabaci is unusually strong after the B-type bemisia tabaci eats the plant with the geminivirus. With the large outbreaks of type B bemisia tabaci, geminiviruses transmitted by bemisia tabaci have caused devastating damage to crops in multiple countries and regions, and have a tendency to grow more severe year by year. For example, in the 90 s of the 20 th century, with the large-scale invasion of the foreign bemisia tabaci in the environment of China, the WTG also spreads to a plurality of provinces, so that the WTG has outbreaks in a plurality of tomato producing areas in 18 province and city autonomous regions at present, the economic loss exceeds 50 hundred million in 2009, the harm is further expanded in 2010-2012, the annual area is more than 100 ten thousand mu, and the WTG becomes an important restriction factor for tomato production in China.

Rice Stripe Virus (RSV), a virus of the genus parvovirus of the family leukoviridae. The virus is only infected by vector insect Laodelphax striatellus, once RSV infects Laodelphax striatellus, the Laodelphax striatellus is finally infected and can continuously transmit the virus. The RSV infected plants are usually withered and have poor pregnancies or small and unrealistic spikes. Yellow green stripes appear at the lower part of the sword leaves after jointing, and various types of rice are not withered, but have abnormal heading and little fructification. Severely restricting the yield of rice.

At present, the prevention and cure measure of insect-borne virus diseases widely used in production is to use chemical insecticide to kill vector insects. However, due to the unreasonable use of chemical drugs by people and the inherent disadvantages of drugs, the problems of environmental pollution, poisoning of people and livestock, pesticide residue and the like are caused when chemical pesticides are used, and with the improvement of the quality of life of people and the enhancement of environmental awareness, public calls for the restriction of chemical pesticides are higher and higher. In order to find a new method for replacing chemical pesticides, people look to safe, stable, pollution-free and efficient natural medicines.

C21-pregnane is widely used in regulating immunity, resisting oxidation, inhibiting acetylcholinesterase, resisting fungi, etc.

Cynanchum otophyllum aglycone (alias: Cynanchum aglycone), English name (Qingyangshenggenin), in this case abbreviated as GSX3, CAS number (84745-94-8), molecular formula (C)₂₈H₃₆O₈) Molecular weight (500.58064), formula (lb) as follows:

disclosure of Invention

The invention aims to provide a novel application of C21-pregnane and derivatives thereof.

The invention provides a new application of C21-pregnane and derivatives thereof, which comprises the following steps: application of C21-pregnane and its derivatives in resisting plant virus is disclosed.

In the application, the C21-pregnane and derivatives thereof have the structural formula shown as follows:

the plant virus includes DNA virus and RNA virus,

the DNA virus may be specifically a geminiviridae virus, which is Begomovirus of the genus Phaseolus aureus, also known as whitefly-transmitted geminivirus (WTG),

the geminiviridae viruses include monocomponent viruses (e.g., Tomato Yellow Leaf Curl Virus (TYLCV)) and bicomponent viruses (e.g., Cassava Mosaic Virus (CMV));

the RNA virus is specifically Rice Stripe Virus (RSV).

The invention also provides a method for preventing and controlling the toxicity of the plant viruses.

The method for preventing and treating the plant virus poison provided by the invention comprises the following steps: c21-pregnane and its derivatives are applied to prevent and treat plant virus poisoning.

The phytovirus poison is caused by a plant virus;

the plant virus includes DNA virus and RNA virus,

the DNA virus may be specifically a geminiviridae virus, which is Begomovirus of the genus Phaseolus aureus, also known as whitefly-transmitted geminivirus (WTG),

the geminiviridae viruses include monocomponent viruses (e.g., Tomato Yellow Leaf Curl Virus (TYLCV)) and bicomponent viruses (e.g., Cassava Mosaic Virus (CMV));

the RNA virus is specifically Rice Stripe Virus (RSV).

According to the invention, geminivirus is inoculated by an agrobacterium injection method, the anti-WTG activity screening is carried out on C21-pregnane monomer compounds derived from plants and derivatives thereof by combining RT-qPCR (reverse transcription-quantitative polymerase chain reaction) to determine the relative virus titer of infected plants, and GSX3 is screened out from the anti-WTG activity screening as a lead compound for resisting geminivirus. By inoculating the double-component geminivirus SLCMV-HN7, the disease of the plant pretreated by the GSX3 is obviously slowed compared with a DMSO control, which indicates that the GSX3 has broad spectrum of resistance to the geminivirus.

The plant-derived antiviral agent GSX3 has the advantages of environmental friendliness, difficulty in drug resistance generation and the like.

Drawings

FIG. 1 shows GSX3 activity against tomato yellow leaf curl virus;

FIG. 2 shows GSX3 activity against cassava mosaic virus;

FIG. 3 shows GSX3 activity against rice stripe virus;

FIG. 4 shows the activity of GSX3 and its derivatives against tomato yellow leaf curl virus.

FIG. 5 shows that GSX3 and its derivatives have anti-cassava mosaic virus activity.

Detailed Description

The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.

The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, biomaterials, etc. used in the following examples are commercially available unless otherwise specified.

GSX2, GSX3 and GSX4 used in the following examples are monomeric compounds extracted, separated and identified from tubers of the traditional Chinese medicine cynanchum wilfordii, and the specific operations are shown in compounds 8,14 and 11 in Huang et al 2015. The derivatives GSX3-10a and GSX3-12 are products synthesized by taking GSX3 as raw materials, and the specific synthetic route is shown in compounds 10c and 10a in Huang et al, 2016.

[1]Huang L.J.,Wang B,Zhang J.X.,et al.Studies on cytotoxic pregnane sapogenins from Cynanchum wilfordii.Fitoterapia,2015,101:107-116.

[2]Huang L.J.,Wang B,Zhang J.X.,et al.Synthesis and evaluation of antifungal activity of C21-steroidal derivatives.Bioorganic&Medicinal Chemistry Letters 2016, 26(8):2040-2043.

Example 1

Selecting a good-growing original tobacco (Nicotiana benthamiana) containing 4-6 leaves with large size; spraying 0.5 mu M GSX3 aqueous solution on leaf surfaces; after 6 hours of treatment, agrobacterium containing tomato yellow leaf curl virus plasmid was injected into tobacco from the back of the leaf by agrobacterium injection method, with DMSO as a negative control solvent. After 10 days, 0.1g of systemic leaf samples of the susceptible plants were weighed, DNA was extracted by CTAB method, and then relative viral titers of the susceptible plants were determined by RT-qPCR using TYLCV primers (see Table 1). FIG. 1 shows GSX3 activity against tomato yellow leaf curl virus; as can be seen from fig. 1: compared to DMSO control, GSX3 pre-treated plants showed significantly reduced leaf curl symptoms (fig. 1A) and also a significant reduction in the relative viral titer of the affected plants (fig. 1B). It shows that GSX3 has the function of protecting the plant against the activity of tomato yellow leaf curl virus.

Example 2

Selecting a good-growing original tobacco (Nicotiana benthamiana) containing 4-6 leaves with large size; spraying 0.5 mu M GSX3 aqueous solution on leaf surfaces; after 6 hours of treatment, agrobacterium containing cassava mosaic virus plasmid was injected into tobacco from the leaf back by agrobacterium injection, solvent DMSO being a negative control, 3 replicates for each 10 plants treated. After 10 days, 0.1g of systemic leaf samples of the susceptible plants were weighed, DNA was extracted by CTAB method, and then relative viral titers of the susceptible plants were determined by RT-qPCR using the SLCMV-HN7 primer (see Table 1). FIG. 2 shows GSX3 activity against cassava mosaic virus; as can be seen from fig. 2: the leaf roll symptoms were significantly reduced in GSX 3-pretreated plants relative to DMSO control (fig. 2A), and qPCR results also showed a significant reduction in virus accumulation in GSX 3-pretreated plants compared to DMSO control (fig. 2B). Shows that GSX3 has the function of protecting the activity of the plant against the cassava mosaic virus.

Example 3

Selecting rice (Nipponbare) seedlings of 2 weeks, spraying 0,0.1,1 and 10 mu M GSX3 aqueous solution on leaf surfaces of the rice seedlings, treating the rice seedlings for 6 hours, adopting a plastic barrel group inoculation method to release 2-3-year Laodelphax striatellus (Laodelphax striatellus) carrying RSV into the rice seedlings, wherein the population density of the Laodelphax striatellus is 10 heads of the plant infected with RSV, removing the Laodelphax striatellus after 3 days, and continuing to culture the Laodelphax striatellus. After the laodelphax striatellus is removed for two weeks, photographing and recording the disease symptoms of the inoculated rice; meanwhile, 0.1g of rice sample is weighed, RNA is extracted by a Trizol kit method, the RNA is reversely transcribed into cDNA by a reverse transcription kit, and then the relative virus titer of the infected plant is determined by RT-qPCR (reverse transcription-quantitative polymerase chain reaction) by an RSV SP primer (shown in table 1).

FIG. 3 shows GSX3 activity against rice stripe virus; as can be seen from fig. 3: after two weeks of inoculation of the RSV on the rice seedlings, the heart leaves of the new rice plants pretreated by the negative control DMSO appear to be dry to a certain degree, and the curled dry leaves at the leaf tips gradually spread downwards along with the passage of time, and the heart leaves die. In contrast, the plant sprayed with GSX3 showed a gradual decrease in the curling and withering of the new leaves with increasing concentration, and when the concentration reached 10. mu.M, the new leaves tended to grow normally in Mock rice (FIG. 3A). The results of the RT-qPCR assay were also consistent with the symptomatic phenotype (fig. 3B). The above results indicate that GSX3 has activity against rice stripe disease virus, and the antiviral activity is concentration dependent.

Example 4

GSX3 and its derivatives were tested for tomato yellow leaf curl virus activity by reference to the procedure of example 1, and the results are shown in FIG. 4.

Example 5

GSX3 and its derivatives were tested for cassava mosaic virus activity by the procedure of example 2, and the results are shown in FIG. 5.

TABLE 1 primers used in the present invention for detecting plant viral titers

Primer and method for producing the same	Sequence (5'-3')	Purpose(s) to
			TYLCV-F	TATGTTAGCTATTAAATATTTGC	RT-qPCR
TYLCV-R	AGCACGGCTGCTGTATGGGC	RT-qPCR
			SLCMV-HN7-F	CACGATGTGGTCCATATAGGTAAG	RT-qPCR
SLCMV-HN7-R	TACGATCCCTTACAAGGAAGAACA	RT-qPCR
			Nb-EF1α-F	TGGTGTCCTCAAGCCTGGTATGGTTG	RT-qPCR
Nb-EF1α-R	ACGCTTGAGATCCTTAACCGCAACATTCTT	RT-qPCR
			RSV-SP-q-F	CTGCCTGAGACTGTTAGCGA	RT-qPCR
RSV-SP-q-R	GTGTCAGTCTCCAAGGGGTG	RT-qPCR
			OsActin2-F	TGCTATGTACGTCGCCATCCAG	RT-qPCR
OsActin2-R	AATGAGTAACCACGCTCCGTC	RT-qPCR