CN116536351A - Genetic engineering application of rice gene ORYsa and SHR2 - Google Patents
Genetic engineering application of rice gene ORYsa and SHR2 Download PDFInfo
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- CN116536351A CN116536351A CN202310414506.6A CN202310414506A CN116536351A CN 116536351 A CN116536351 A CN 116536351A CN 202310414506 A CN202310414506 A CN 202310414506A CN 116536351 A CN116536351 A CN 116536351A
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- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 47
- 235000009566 rice Nutrition 0.000 title claims abstract description 47
- 101150009276 SHR2 gene Proteins 0.000 title claims abstract description 38
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- 238000010353 genetic engineering Methods 0.000 title claims abstract description 7
- 240000007594 Oryza sativa Species 0.000 title 1
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- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 33
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 33
- 239000011574 phosphorus Substances 0.000 claims abstract description 33
- 238000010521 absorption reaction Methods 0.000 claims abstract description 15
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Abstract
The invention discloses a rice gene ORYsa; genetic engineering application of SHR2. The gene is applied to improving the phosphorus absorption efficiency in soil and enhancing the tillering occurrence capacity of rice. The gene of the invention is the first report on the phosphorus absorption in rice, and plays an important role in improving the phosphorus absorption efficiency in soil. ORYsa; after SHR2 is knocked out, the phosphorus absorption efficiency can be improved by 29% -48%, the capacity of rice tillering is improved, and a guarantee is provided for cultivating new rice varieties suitable for phosphorus-lean soil.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a rice gene ORYsa; genetic engineering application of SHR2.
Background
Phosphorus (P) is a macronutrient element necessary for plant growth and development, and is involved in plant photosynthesis, substance metabolism, and energy transfer in addition to being an important component of biomacromolecules such as nucleic acids, ATP, phospholipids, and the like in plants (ragkothama, 1999;Poirier and Bucher,2002;L mez-arondo et al, 2014). Phosphorus in natural soil is very inefficient and is lacking for most crop production activities (Bieleski, 1973). Moreover, the crop has extremely low utilization rate of phosphate fertilizer, and the low utilization rate leads to the necessity of applying a large amount of phosphate fertilizer in agricultural production; phosphate rock used for producing phosphate fertilizer in nature is a non-renewable resource, and scientists expect that low-cost phosphate fertilizer resources will be exhausted in about 2050 (Vance et al, 2003), besides, phosphate fertilizer can flow into other ecological systems through processes of leaching, water movement and the like, so that a series of ecological environment problems such as water eutrophication and the like are caused (L motion-Arradndo et al, 2014; liu et al, 2016). So that the current phosphorus deficiency of soil and surplus phosphate fertilizer application become one of key factors for limiting crop yield, resource waste and environmental destruction.
SHR is a plant-specific transcription factor protein, and is found to be expressed in the center pillar cells of arabidopsis roots, and the protein can move from the center pillar cells to adjacent endothelial layer cells, so that the expression of attsc in the layer of cells is activated, and finally, the expression of attsc and the attsc protein are used for regulating and controlling the asymmetric pericycle division process of the root system cells of arabidopsis (helaritta et al, 2000;Nakajima et al, 2001). Although specific expression of the SHR-SCR module in the endothelial layer is a molecular guarantee that the endothelial layer and the cortical cell differentiate, in recent researches, it has been found that the SHR-SCR module in leguminous plants can be specifically expressed in the cortical cell, and the specific expression has a close relationship with nitrogen-fixing bacteria symbiosis, which is a molecular basis that leguminous plant root systems and nitrogen-fixing bacteria can symbiosis (Dong et al, 2020), and further researches show that GmSHR4/5 directly activates downstream D-type cyclin (GmCYCD 6; 1-6) to form a feed-forward loop, thereby regulating a key mechanism of initiating division of soybean rhizobium primordium (Wang et al, 2021). However, the prior art does not disclose ORYsa; effect of SHR2 on phosphorus uptake and rice tillering.
Disclosure of Invention
The invention aims to provide a rice transcription factor gene ORYsa; the gene engineering application of SHR2, the gene knockout in rice improves the phosphorus absorption and utilization of the rice and promotes the rice tillering.
The aim of the invention can be achieved by the following technical scheme:
rice transcription factor gene ORYsa; the genetic engineering application of SHR2 is that a rice transcription factor gene ORYsa is knocked out from rice; SHR2 can improve rice phosphorus absorption and utilization, promote rice tillering and/or reduce plant height, and the rice transcription factor gene ORYsa; the gene sequence accession number of SHR2 at NCBI is XM_015773807.2 (https:// www.ncbi.nlm.nih.gov/nuccore/XM_015773807.2 report=fasta).
Knock out or silence ORYsa; application of SHR2 gene substances in improving rice phosphorus absorption and utilization, promoting rice tillering and/or reducing plant height.
As a preferred aspect of the invention, the knockout or silencing orisa; the substance of the SHR2 gene is directed against ORYsa; CRISPR/Cas9 gene knockout system of SHR2 gene, or ORYsa; siRNA of SHR2 gene.
Advantageous effects
The gene ORYsa of the invention; the SHR2 has the function of first report in rice phosphorus absorption and plays an important role in improving the phosphorus absorption and utilization efficiency in soil. ORYsa; SHR2 is taken as a target gene to be knocked out in rice, and mutation of the SHR2 can improve phosphorus absorption, improve the absorption rate of a rice root system to phosphorus, promote rice tillering, reduce the plant height of the rice and provide a guarantee for cultivating new rice varieties suitable for phosphorus-deficient soil.
Drawings
FIG. 1 spatial and temporal expression pattern of OsSHR2 Gene
Fig. 2.Orysa; identification of expression pattern of SHR2 Gene under the absence of element
FIG. 3 ORYsa; SHR2 mutant and wild rice aerial and underground dry weight
Fig. 4.Orysa; total phosphorus content concentration of SHR2 mutant and wild rice plant parts
Fig. 5.Orysa; SHR2 mutant and wild rice plant height and tillering character
RYSa, FIG. 6; SHR2 mutant and wild rice total phosphorus concentration at different parts of flowering stage and mature stage
Fig. 7.Orysa; SHR2 gene editing vector
Detailed Description
Example 1 acquisition of Gene sequences
Applicants input XM 015773807.2 on NCBI website (www.ncbi.nlm.nih.gov) to obtain a DNA sequence encoding GRAS family transcription factor gene.
Example 2, ORYsa; expression Pattern identification of SHR2
2.1 extraction of Total RNA and reverse transcription to synthesize first strand cDNA
Selecting rice variety Nippon-Qing, culturing with complete nutrient solution for 1 week after rice seedling grows to 10 days, treating with different nutrient solutions for 1 week, and sampling. Samples of different parts are taken at different growth periods of the rice. Extracting total RNA by using TriZol reagent, identifying total RNA quality by agarose gel electrophoresis, and then measuring RNA content on a spectrophotometer. And (3) taking the obtained total RNA as a template, and obtaining the rice cDNA through reverse transcription for subsequent experiments. The first strand cDNA synthesis procedure was performed according to the HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wind) kit instructions from Norpraise.
2.2, ORYsa; tissue-specific expression pattern identification of SHR2 genes
And 2, designing the following specific primers P1 and P2 by taking the 'Japanese sunny' cDNA obtained in the step 2.1 as a template according to the coding sequence of the rice OsSHR2 gene, amplifying a 136bp product, and identifying the expression mode of the OsSHR2 gene.
P1 GCAAATCATCGAGCCTGCG
P2 CGTACGTTGCTGGCTGAAAAA
The PCR specific steps were performed according to the instructions of HiScript II Q RT SuperMix for qPCR kit produced by Norwegian; the instrument used was the Quantum studio 6Pro Real-Time PCR System (Thermo FisherScientific) available from the laboratory. OsActin1 gene as an internal referenceBecause of this. Quantitative results according to 2 -ΔC the t method calculates and analyzes the space-time expression pattern of the OsSHR2 gene, and the result is shown in figure 1.
As can be seen from fig. 1, osSHR2 is mainly expressed in roots during vegetative growth phase, whereas expression is higher in stems, seed organs during reproductive growth phase.
2.3, ORYsa; identification of expression pattern of SHR2 Gene under the absence of element
Total RNA extraction, reverse transcription, and fluorescence quantitative PCR methods are the same as 2.1 and 2.2. Is used for detecting the change of the gene expression of OsSHR2 under different time and nutrient deficiency conditions. As shown in FIG. 2, the up-regulation of the expression level of OsSHR2 was most remarkable in the case of phosphorus deficiency.
Example 3, knock-out of ORYsa using CRISPR-Cas9 technology; application prospect of OsSHR2
3.1, ORYsa; SHR2 target selection and design
The PAM (protospacer-adjacent motif) prosequence recognized by CRISPR-Cas9 is NGG. The genome sequence and the gene ID number of the OsSHR2 are searched through a Rice Genome Annotation Project website (http:// price. Plan. Msu. Edu /), and an alternative PAM (15 bp) corresponding to the gene number is firstly found in UltraEdit software, and a proper spacer is screened and designed. When designing the primer, the upstream and downstream primers are respectively added with enzyme cutting sites at the 5' end, and the primer sequences required by constructing the mutant vector are as follows.
S1F:GGCATTCCTCCCGCCAGTTCCACT
S1R:AAACAGTGGAACTGGCGGGAGGAA
S2F:GGCAGTTCCGTCGTCGTCCGGTGC
S2R:AAACGCACCGGACGACGACGGAAC
S3F:GGCACGCCAGTTCCACTCGGGAAC
S3R:AAACGTTCCCGAGTGGAACTGGCG
3.2, ORYsa; construction of SHR2 mutant
Each primer in 3.1 was synthesized and annealed, and the BsaI-sgRNA vector was digested with T4 ligase, and transformed into E.coli DH 5. Alpha. With U3Pro digested with BsaI. The sgRNA and spacer sequences on the pOs-sgRNA vector were replaced into the ph-Ubi-cas9-7 expression vector (FIG. 7) using Gateway technology, and the vector transformed into E.coli DH 5. Alpha. Sequencing the vector (Suzhou Jin Weizhi company), and preserving the escherichia coli for later use after the sequencing is correct.
3.3 acquisition and identification of mutant transgenic plants
The final vector constructed in 3.2 was shock transformed into agrobacterium EHA 105. Then infecting the mature embryo callus of the japonica rice variety Dongjin (jeoneget al, 2006), and then carrying out hygromycin screening and redifferentiation to obtain a positive plant preliminarily. After the mutant T0 generation material is obtained, the DNA of each strain of the T0 generation is extracted, and the target peripheral sequence is amplified by using a specific primer and sequenced and identified (the result is not provided).
3.4, growing conditions of mutant transgenic plants under different phosphorus treatment conditions of the plants
Selecting mutant strain of different strains and seedling of wild rice for 10 days, culturing in complete nutrient solution for 1 week, and transferring HP (200 μm PO 4 3- Normal treatment concentration) nutrient solution and LP (10 μm PO 4 3- ) And (3) after the nutrient solution is treated for two weeks, observing and photographing, and measuring the dry weights of the overground part and the underground part after drying. As shown in fig. 3, the mutant plants under both conditions were significantly reduced compared to the wild-type biomass, either above-ground or below-ground.
3.5, phosphorus concentration of mutant transgenic plants under different phosphorus treatment conditions due to plants
The rice samples of 3.4 were taken and H was used 2 SO 4 -H 2 O 2 After digestion, the total phosphorus concentration of each part of the plant was determined using a flow analyzer. The experimental results are shown in FIG. 4, and under HP conditions, the total phosphorus concentration of both the aerial and underground parts of the mutant was significantly higher than that of the wild type. The phosphorus uptake efficiency was calculated to be significantly higher for the mutant strain than for the wild type under HP conditions.
3.6, agronomic traits of mutant transgenic plants due to plant field conditions
The mutant strains of different strains and the wild rice communities are selected to be planted in the rice field, and the plant height tillering characters of the rice of the different strains are counted in the mature period. As shown in fig. 5, the plant height of the mutant was significantly lower than that of the wild type under small field conditions, and the effective tiller number was significantly increased.
3.7, total phosphorus concentration of mutant transgenic plants due to plant field conditions
The flowering period and the maturity period of the rice planted in 3.6 are sampled respectively. Is divided into 5 parts of stems, old leaves, new leaves, leaf sheaths and seeds. The total phosphorus concentration was determined as indicated in 3.6. The results are shown in FIG. 6, the total phosphorus concentration of the mutant is significantly higher than that of the wild type in the stalks, leaf sheaths and new leaves in the flowering stage; while at maturity the total phosphorus concentration in the stems is still significantly higher than in the wild type.
In summary, the present inventors provide an ORYsa; the engineering application of SHR2 is first reported in rice. ORYsa; the knockout of SHR2 can improve the plant phosphorus absorption rate and promote rice tillering, and provides a guarantee for cultivating new rice varieties with high phosphorus absorption and improved plant types.
For simplicity, ORYsa is used in the present invention; SHR2 is sometimes denoted OsSHR2.
Claims (3)
1. Rice transcription factor gene ORYsa; the genetic engineering application of SHR2 is characterized in that a rice transcription factor gene ORYsa is knocked out in rice; SHR2 can improve rice phosphorus absorption and utilization, promote rice tillering and/or reduce plant height, and the rice transcription factor gene ORYsa; the gene sequence accession number of SHR2 at NCBI is XM_015773807.2.
2. Knock out or silence ORYsa; application of SHR2 gene substances in improving rice phosphorus absorption and utilization, promoting rice tillering and/or reducing plant height.
3. The use according to claim 2, characterized in that said knockout or silencing ORYsa; the substance of the SHR2 gene is directed against ORYsa; CRISPR/Cas9 gene knockout system of SHR2 gene, or ORYsa; siRNA of SHR2 gene.
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