Method for regulating over-ground gene expression of plant by modifying long-distance moving sRNA precursor and application of method

Abstract

The invention discloses a method for regulating and controlling plant overground gene expression by modifying a long-distance moving sRNA precursor and application, and relates to the technical field of plant overground gene expression, and the key point of the technical scheme is as follows: the method realizes regulation and control of plant overground gene expression by firstly screening long-distance moving sRNA and then modifying the long-distance moving sRNA precursor. According to the method, through a series of identified sRNAs which can be moved to the ground from the underground in a long distance, after precursors of the sRNAs are modified, the sRNAs which can be moved to the ground and target the ground gene can be generated through a precursor sequence which is subjected to overexpression modification through simple root agrobacterium tumefaciens transient conversion underground, so that the expression quantity of the ground gene is influenced.

Description

Technical Field

[0001] The present invention relates to the technical field of aboveground gene expression in plants, and more specifically, to a method and application of modifying long-distance mobile sRNA precursors to regulate aboveground gene expression in plants. Background Art

[0002] Small RNA (sRNA) is a class of non-coding RNA with a length between 18 and 30 nucleotides (nt), which are mainly divided into siRNA and microRNA. sRNA plays an important role in regulating plant growth and development, plant-microbe interactions, disease resistance and abiotic stress mitigation. During the biosynthesis of sRNA, Dicer-like protein (DCL) as an RNase III enzyme plays an important role in processing double-stranded RNA (dsRNA) into 20-24 nucleotide siRNA or miRNA. The genome of higher plants encodes at least four Dicer-like (DCL) proteins, DCL1 is mainly responsible for the production of miRNA, while DCL2 is mainly involved in the production of 22-nt secondary siRNA and plays a role in antiviral defense. DCL3 is associated with the formation of 24-nt sRNA associated with RNA-mediated DNA methylation, and DCL4 is mainly involved in the processing of endogenous RDR6-dependent trans-acting siRNA (tasiRNA) and regulates vegetative phase changes. In higher plants, sRNAs move over long distances through the vascular system and participate in gene regulation and epigenetic modification.

[0003] By grafting the wild type and dcl2 mutant, some sRNAs that depend on DCL2 protein synthesis and can move from underground to above ground in the plant were screened, and some of these sRNAs were identified. It was found that the sRNA could move to the ground after overexpressing its precursor sequence underground. After modifying the mature sequence in the precursor, it was found that the modified sequence can still produce sRNA that can move over long distances and can specifically target genes expressed above ground.

[0004] Most existing methods for changing gene expression in plants are through gene editing, which uses transgenic technology to transfer gene knockout or overexpression vectors into plants to regulate gene expression. Transient transformation of plants mediated by Agrobacterium can produce transgenic roots in the roots of plants, changing the gene expression in the transgenic roots. Changing the expression of certain genes in other parts of the plant is mostly done through stable genetic transformation of Agrobacterium, transferring into binary vectors, and integrating special elements in the vector into the plant genome, thereby changing gene expression in the plant.

[0005] However, existing experimental technologies make it difficult to change the gene expression of the aboveground part of the plant within a short experimental period without destroying the plant. Existing gene editing technologies are achieved through Agrobacterium-mediated root transient transformation experiments or stable genetic transformation, but root transient transformation cannot change the gene expression in other parts of the plant, and stable genetic transformation is time-consuming and costly. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application for modifying long-distance mobile sRNA precursors to regulate the expression of aboveground genes in plants. The method provided by the present invention identifies a series of sRNAs that can move long distances from underground to aboveground, and then modify their precursors and then overexpress the modified precursor sequences underground through simple root Agrobacterium transient transformation, which can produce some sRNAs that can move to the ground and target aboveground genes, thereby affecting the expression level of aboveground genes.

[0007] The above technical objectives of the present invention are achieved through the following technical solutions: a method for modifying long-distance mobile sRNA precursors to regulate aboveground gene expression in plants, the method comprising the following two steps:

[0008] S1. Screening of long-distance mobile sRNAs;

[0009] S2. Modification of long-distance mobile sRNA precursors.

[0010] The present invention is further configured as follows: the specific steps of screening the long-distance mobile sRNA in S1 are as follows:

[0011] (1) By comparing the sRNAs measured above and below ground in different grafted materials, the sRNAs that depend on the production of DCL2 protein in the wild type were screened out;

[0012] (2) Screening of grafted materials: wild type above ground and wild type underground ( WT |WT) and the aboveground is wild type and the underground is mutant ( WT |dcl2) was detected above ground and was a mutant above ground and also a mutant underground ( dcl2 |dcl2) sRNAs that do not exist on the ground, these sRNAs are the sRNAs that are dependent on the DCL2 protein production on the wild-type ground;

[0013] (3) Screening of grafted materials that are wild type above ground and wild type underground (WT| WT ) and aboveground is mutant and underground is wild type (dcl2| WT ) detected underground and above is a mutant underground is also a mutant (dcl2| dcl2 ) sRNAs that do not exist in the underground, these sRNAs are the sRNAs that are produced in the wild-type underground and are dependent on the DCL2 protein;

[0014] (4) integrating these sRNAs together to obtain a library of all sRNAs that are dependent on DCL2 protein production in the wild type;

[0015] (5) Then the mutant is above ground and the wild type is underground ( dcl2 |WT) aboveground materials to identify which sRNAs are common in the sRNA library that depends on DCL2 protein production in the wild type, that is, sRNAs that depend on DCL2 protein production in the wild type and can move from underground to aboveground can be screened.

[0016] The present invention is further configured as follows: the sRNA library includes: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70.

[0017] The present invention is further configured as follows: the specific steps of transforming the long-distance mobile sRNA precursor are as follows:

[0018] 200 bp before and after the sRNA sequence that can move from underground to above ground screened out in S1 were selected as its precursor sequence, and its precursor sequence was cloned into a root overexpression vector. The roots were transiently transformed with K599 Agrobacterium to overexpress the sRNA precursor in the roots.

[0019] The present invention also provides application of the above method in regulating the aboveground gene expression of plants.

[0020] In summary, the present invention has the following beneficial effects:

[0021] 1. The present invention screened a series of DCL2-dependent sRNA sequences that can move from underground to aboveground by using wild-type and mutant grafting.

[0022] 2. The present invention designs primers to modify the precursor sequences of these sRNAs, which can produce sRNAs targeting specific sequences after being overexpressed underground, and can move over long distances to the ground to target and inhibit the expression of target genes in the aboveground part.

[0023] 3. The present invention can utilize a series of mobile sRNAs discovered, and after designing experiments to modify their precursor sequences, it can influence the expression level of target genes in the aboveground part within a short test period through simple root transformation experiments, thus providing an effective technical means for phenotypic prediction, gene function verification, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the method for screening sRNAs that are dependent on the production of DCL2 protein in the wild type in the embodiment of the present invention;

[0025] Figure 2 In the embodiment of the present invention, sRNA that can move from underground to above ground in a DCL2-dependent manner in the wild type is screened;

[0026] Figure 3 The sRNA precursor in the embodiment of the present invention is connected to the root transfection expression vector;

[0027] Figure 4 These are the results of qRT-PCR testing of various tissues in the sRNA precursor root overexpression experiment in the embodiment of the present invention;

[0028] Figure 5 This is the principle of sRNA precursor modification targeting above-ground genes in the embodiments of the present invention;

[0029] Figure 6 It is the detection of aboveground gene expression in each tissue after the sRNA precursor is modified in the embodiment of the present invention;

[0030] Figure 7 It is the efficiency of the expression of above-ground genes after the modification of different sRNA precursors in the embodiments of the present invention. DETAILED DESCRIPTION

[0031] The following is combined with Figure 1-7 The present invention is described in further detail.

[0032] Example: Method and application of modifying long-distance mobile sRNA precursors to regulate aboveground gene expression in plants

[0033] 1. Screening of long-distance mobile sRNA

[0034] By comparing the sRNAs measured above and below ground in different grafted materials, sRNAs that depend on the production of DCL2 protein in the wild type were screened out; sRNAs that are wild type above ground and wild type underground were screened out. WT |WT) and the aboveground is wild type and the underground is mutant ( WT |dcl2) was detected above ground and was a mutant above ground and also a mutant underground ( dcl2 |dcl2) sRNAs that do not exist above ground, these sRNAs are the sRNAs produced by the wild type above ground and dependent on DCL2 protein; screening of grafted materials that are wild type above ground and wild type underground (WT| WT ) and aboveground is mutant and underground is wild type (dcl2| WT ) detected underground and above is a mutant underground is also a mutant (dcl2| dcl2 ) sRNAs that do not exist underground, these sRNAs are the wild-type sRNAs that are dependent on DCL2 protein production underground; these sRNAs are integrated together to obtain a library of sRNAs that are dependent on DCL2 protein production in all wild-types; then the mutants are above ground and the wild-type is underground ( dcl2 |WT) aboveground materials to identify which sRNAs are common in the sRNA library that depends on DCL2 protein production in the wild type, that is, sRNAs that depend on DCL2 protein production in the wild type and can move from underground to aboveground can be screened.

[0035] Table 1. List of DCL2-dependent sRNAs that can move from underground to aboveground in wild type

[0036]

[0037]

[0038]

[0039] The above-mentioned DCL2-Mob-sRNA-1 sequence is SEQ ID NO: 1, the DCL2-Mob-sRNA-2 sequence is SEQ ID NO: 2, the DCL2-Mob-sRNA-3 sequence is SEQ ID NO: 3, the DCL2-Mob-sRNA-4 sequence is SEQ ID NO: 4, the DCL2-Mob-sRNA-5 sequence is SEQ ID NO: 5, the DCL2-Mob-sRNA-6 sequence is SEQ ID NO: 6, the DCL2-Mob-sRNA-7 sequence is SEQ ID NO: 7, the DCL2-Mob-sRNA-8 sequence is SEQ ID NO: 8, the DCL2-Mob-sRNA-9 sequence is SEQ ID NO: 9, the DCL2-Mob-sRNA-10 sequence is SEQ ID NO: 10, the DCL2-Mob-sRNA-11 sequence is SEQ ID NO: 11, and the DCL2-Mob-sRNA-12 sequence is SEQ ID NO: 13. NO: 12, DCL2-Mob-sRNA-13 sequence is SEQ ID NO: 13, DCL2-Mob-sRNA-14 sequence is SEQ ID NO: 14, DCL2-Mob-sRNA-15 sequence is SEQ ID NO: 15, DCL2-Mob-sRNA-16 sequence is SEQ ID NO: 16, DCL2-Mob-sRNA-17 sequence is SEQ ID NO: 17, DCL2-Mob-sRNA-18 sequence is SEQ ID NO: 18, DCL2-Mob-sRNA-19 sequence is SEQ ID NO: 19, DCL2-Mob-sRNA-20 sequence is SEQ ID NO: 20, DCL2-Mob-sRNA-21 sequence is SEQ ID NO: 21, DCL2-Mob-sRNA-22 sequence is SEQ ID NO: 22, and DCL2-Mob-sRNA-23 sequence is SEQ ID NO: 23. NO:23, DCL2-Mob-sRNA-24 sequence is SEQ ID NO:24, DCL2-Mob-sRNA-25 sequence is SEQ ID NO:25, DCL2-Mob-sRNA-26 sequence is SEQ ID NO:26, DCL2-Mob-sRNA-27 sequence is SEQ ID NO:27, DCL2-Mob-sRNA-28 sequence is SEQ ID NO:28, DCL2-Mob-sRNA-29 sequence is SEQ ID NO:29, DCL2-Mob-sRNA-30 sequence is SEQ ID NO:30, and DCL2-Mob-sRNA-31 sequence is SEQ ID NO:31,The DCL2-Mob-sRNA-32 sequence is SEQ ID NO:32, the DCL2-Mob-sRNA-33 sequence is SEQ ID NO:33, the DCL2-Mob-sRNA-34 sequence is SEQ ID NO:34, the DCL2-Mob-sRNA-35 sequence is SEQ ID NO:35, the DCL2-Mob-sRNA-36 sequence is SEQ ID NO:36, the DCL2-Mob-sRNA-37 sequence is SEQ ID NO:37, the DCL2-Mob-sRNA-38 sequence is SEQ ID NO:38, the DCL2-Mob-sRNA-39 sequence is SEQ ID NO:39, the DCL2-Mob-sRNA-40 sequence is SEQ ID NO:40, the DCL2-Mob-sRNA-41 sequence is SEQ ID NO:41, the DCL2-Mob-sRNA-42 sequence is SEQ ID NO:42, and the DCL2-Mob-sRNA-43 sequence is SEQ ID NO:43. ID NO:43, DCL2-Mob-sRNA-44 sequence is SEQ ID NO:44, DCL2-Mob-sRNA-45 sequence is SEQ ID NO:45, DCL2-Mob-sRNA-46 sequence is SEQ ID NO:46, DCL2-Mob-sRNA-47 sequence is SEQ ID NO:47, DCL2-Mob-sRNA-48 sequence is SEQ ID NO:48, DCL2-Mob-sRNA-49 sequence is SEQ ID NO:49, DCL2-Mob-sRNA-50 sequence is SEQ ID NO:50, DCL2-Mob-sRNA-51 sequence is SEQ ID NO:51, DCL2-Mob-sRNA-52 sequence is SEQ ID NO:52, DCL2-Mob-sRNA-53 sequence is SEQ ID NO:53, DCL2-Mob-sRNA-54 sequence is SEQ ID NO:54. NO:54, DCL2-Mob-sRNA-55 sequence is SEQ ID NO:55, DCL2-Mob-sRNA-56 sequence is SEQ ID NO:56, DCL2-Mob-sRNA-57 sequence is SEQ ID NO:57, DCL2-Mob-sRNA-58 sequence is SEQ ID NO:58, DCL2-Mob-sRNA-59 sequence is SEQ ID NO:59, DCL2-Mob-sRNA-60 sequence is SEQ ID NO:60, DCL2-Mob-sRNA-61 sequence is SEQ ID NO:61, and DCL2-Mob-sRNA-62 sequence is SEQ ID NO:62,The sequence of DCL2-Mob-sRNA-63 is SEQ ID NO:63, the sequence of DCL2-Mob-sRNA-64 is SEQ ID NO:64, the sequence of DCL2-Mob-sRNA-65 is SEQ ID NO:65, the sequence of DCL2-Mob-sRNA-66 is SEQ ID NO:66, the sequence of DCL2-Mob-sRNA-67 is SEQ ID NO:67, the sequence of DCL2-Mob-sRNA-68 is SEQ ID NO:68, the sequence of DCL2-Mob-sRNA-69 is SEQ ID NO:69, and the sequence of DCL2-Mob-sRNA-70 is SEQ ID NO:70.

[0040] 2. sRNA root transfection expression detection

[0041] The soybean genome information website was used to search for DCL2-dependent sRNA sequences that could move from underground to aboveground. 200 bp before and after the sRNA sequences were selected as their precursor sequences, and the precursor sequences were cloned into a root overexpression vector. The roots were transiently transformed with K599 Agrobacterium to overexpress the sRNA precursors in the roots. Then, aboveground and underground tissues were collected and compared with the transformed empty vector for t-test. The expression levels of sRNA in each tissue were detected by qRT-PCR. It was found that after most of the sRNA precursors were overexpressed underground, the expression levels of the aboveground sRNAs were also significantly increased, indicating that sRNA can indeed move to the ground with high efficiency.

[0042] Select the sRNA with significantly increased sRNA expression in the aboveground part after precursor root transfection in the verification results, and artificially transform the sRNA mature sequence in its precursor into a complementary sequence of the aboveground expressed gene, in order to target the gene through sequence complementarity to affect its expression. Sequence modification introduces mismatch sequences by designing primers, and then replaces the new sequence into the original root transfection expression vector through sequence overlap, that is, the original precursor sequence can be used to produce the designed sRNA.

[0043] The modified sRNA precursor may target the expression level of the aboveground target gene. The modified sRNA precursor sequence was overexpressed in the underground root transformation experiment, and then the expression level of sRNA in various aboveground and underground tissues was detected. It was found that the expression level of some aboveground target genes was significantly inhibited, indicating that the modified precursor sequence can produce sRNA targeting the target gene and can move to the ground to inhibit the expression of the target gene.

[0044] In the specific implementation process, we selected some mobile sRNAs for verification and found that multiple sRNAs can be successfully overexpressed underground and then moved to the ground. After modifying the mature sequences of some of the sRNAs, sRNAs that can target aboveground genes were produced and the expression levels of aboveground genes were changed. For example, after the DCL2-Mob-sRNA-28 precursor was modified, the expression levels of the aboveground expressed genes Glyma.13G276700, Glyma.06G207800, Glyma.13G232500 and other genes were detected by qRT-PCR. After the T test calculation, it was found that the expression levels in some root-transformed plants were significantly inhibited compared with the empty vector control. After the precursors such as DCL2-Mob-sRNA-52 and DCL2-Mob-sRNA-69 were modified, it was also found that some root-transformed plants significantly inhibited the expression levels of the aboveground expressed genes Glyma.13G276700, Glyma.06G207800, and Glyma.13G232500.

[0045] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for modifying long-distance mobile sRNA precursors to regulate aboveground gene expression in plants, characterized in that: The method comprises the following two steps: S1. Screening of long-distance mobile sRNAs; S2. Modification of long-distance mobile sRNA precursors.

2. The method for modifying long-distance mobile sRNA precursors to regulate aboveground gene expression in plants according to claim 1, characterized in that: The specific steps of screening long-distance mobile sRNA in S1 are as follows: (1) By comparing the sRNAs measured above and below ground in different grafted materials, the sRNAs that depend on the production of DCL2 protein in the wild type were screened out; (2) Screening of grafted materials: wild type above ground and wild type underground ( WT |WT) and the aboveground is wild type and the underground is mutant ( WT |dcl2) was detected above ground and was a mutant above ground and also a mutant underground ( dcl2 |dcl2) sRNAs that do not exist on the ground, these sRNAs are the sRNAs that are dependent on the DCL2 protein production on the wild-type ground; (3) Screening of grafted materials that are wild type above ground and wild type underground (WT| WT ) and aboveground is mutant and underground is wild type (dcl2| WT ) detected underground and above is a mutant underground is also a mutant (dcl2| dcl2 ) sRNAs that do not exist in the underground, these sRNAs are the sRNAs that are produced in the wild-type underground and are dependent on the DCL2 protein; (4) integrating these sRNAs together to obtain a library of all sRNAs that are dependent on DCL2 protein production in the wild type; (5) Then the mutant is above ground and the wild type is underground ( dcl2 |WT) aboveground materials to identify which sRNAs are common in the sRNA library that depends on DCL2 protein production in the wild type, that is, sRNAs that depend on DCL2 protein production in the wild type and can move from underground to aboveground can be screened.

3. The method for modifying long-distance mobile sRNA precursors to regulate aboveground gene expression in plants according to claim 2, characterized in that: The sRNA library includes: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:

70.

4. The method for modifying long-distance mobile sRNA precursors to regulate aboveground gene expression in plants according to claim 3, characterized in that: The specific steps for modifying the long-distance mobile sRNA precursor are as follows: 200 bp before and after the sRNA sequence that can move from underground to above ground screened out in S1 were selected as its precursor sequence, and its precursor sequence was cloned into a root overexpression vector. The roots were transiently transformed with K599 Agrobacterium to overexpress the sRNA precursor in the roots.

5. Use of the method according to any one of claims 1 to 4 in regulating aboveground gene expression in plants.

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