CN116804200B - Normal wheat tillering angle control gene TaTB1-2-A and protein encoded by same - Google Patents
Normal wheat tillering angle control gene TaTB1-2-A and protein encoded by same Download PDFInfo
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Abstract
The invention belongs to the field of genetic engineering, and discloses a common wheat tillering angle control gene TaTB1-2-A and a protein encoded by the same. cDNA sequence of TaTB1-2-A gene from Wangshuibai is shown as SEQ ID NO:1, the coding amino acid sequence of the TaTB1-2-A gene is shown as SEQ ID NO: 3. The high expression of the TaTB1-2-A gene has the effect of obviously increasing the tillering angle in wheat, so that the gene can be combined with an over-expression promoter in plants, then introduced into a proper expression vector and transformed into a plant host, further the tillering angle of the plants is changed, and the yield of the plants is regulated.
Description
Technical Field
The invention belongs to the field of genetic engineering, and relates to a common wheat tillering angle control gene TaTB1-2-A and a protein encoded by the same.
Background
Wheat (triticum aestinum l.) is the grain crop with the largest planting area worldwide, and can supply more than 35% of total human population worldwide, providing various proteins, minerals and vitamins for human beings, wherein about 20% of total calories consumed by humans are provided by wheat. China is the largest wheat producer and consumer world in the world, and the wheat yield accounts for about 17% of the total global yield. With the increasing population, it is expected that grain production will need to increase by 70% -100% before 2050 to meet human needs. Therefore, ensuring the safe production of wheat is directly related to the grain safety and social stability of China.
Since the last 70 th century, the world grain yield is almost doubled due to the use of rice semi-dwarf gene SD1 and wheat Rht1 gene, the world grain safety problem is basically solved, and the green revolution becomes a classical case for improving the crop yield by utilizing plant types. In recent years, high-yield wheat breeding encounters new bottlenecks, and negative effects such as reduction of light energy utilization rate caused by dense overlapping of leaf layers and the like are brought about along with reduction of wheat plant height. The wheat tillering is one of important characters of plant types, and reasonable tillering angles can not only increase sowing density, but also improve photosynthetic efficiency of groups and resistance of plants, thereby influencing yield and quality of wheat. In the south-north transition zone of China province, the climate is changeable, and particularly, the climate in the Huainan region is warm and moist, and the scab is frequently repeated due to more rainwater in the middle and later periods of wheat growth. The sowing density directly influences the windability and the light permeability of wheat Tian Tong, so that the damage caused by scab can be reduced due to a reasonable plant type structure. Therefore, the method for excavating the wheat tillering angle regulation genes and exploring the mechanism of wheat plant type regulation not only can solve the basic problem in plant science, but also has important practical significance for breeding of high-yield high-quality wheat new varieties.
The tillering angle refers to the included angle between the side tillers and the main stem, and is an important component factor of plant type. Plants can be classified into three types according to the size of the tillering angle: bunching (plant tillering angle less than 20 °), compacting (plant tillering angle between 21-32 °) and loosening (plant tillering angle greater than 33 °). The tillering angle is a complex agronomic character, is closely related to the yield of crop groups, and is comprehensively regulated and controlled by heredity, hormone, environment and the like. In the plant type research of crops such as wheat, rice and the like, the research of tillering angles is always a hot spot and a difficult point. Therefore, the positioning, cloning and molecular mechanism analysis of the gene controlling the tillering angle have important theoretical significance and application value for improving the plant type of crops and further improving the yield.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing a gene TaTB1-2-A related to the tillering angle of wheat.
It is another object of the present invention to provide the use of the gene.
The aim of the invention can be achieved by the following technical scheme:
a gene TaTB1-2-A from the Wangshui white related to the tillering growth of wheat, wherein the cDNA nucleotide sequence of the gene TaTB1-2-A from the Wangshui white is shown as SEQ ID NO: 1.
CDS of the gene TaTB1-2-A is shown as SEQ ID NO: 2.
The amino acid sequence of the gene TaTB1-2-A is shown as SEQ ID NO: 3.
The TaTB1-2-A can be synthesized artificially or can be obtained by synthesizing the coding gene and then biologically expressing.
The DNA sequences defined above have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology and encode DNA molecules having the same functional protein.
Genes encoding proteins of the above-mentioned similar domains in other plants are also within the scope of the present invention.
The gene TaTB1-2-A related to the tillering angle of the wheat, and the expression cassette, the recombinant vector or the recombinant microorganism which are derived from the gene TaTB1-2-A and contain the gene TaTB1-2-A.
The gene TaTB1-2-A is applied as follows (A1) or (A2): (A1) controlling the tillering angle of a plant; (A2) enhancing or reducing the tillering angle of the plant.
The plant is monocotyledonous plant or dicotyledonous plant; the monocot plant may be a gramineous plant.
The use of the expression cassette, recombinant vector or recombinant microorganism is characterized by the following (B1) or (B2): the transgenic plant with changed tillering angle cultivated in the step (B1); (B2) Transgenic plants with increased or decreased tillering angle are cultivated.
The plant is monocotyledonous plant or dicotyledonous plant; the monocot plant may be a gramineous plant.
The beneficial effects are that:
the high expression of the TaTB1-2-A gene has the effect of obviously increasing the tillering angle in wheat, and the TaTB1-1-D with 42.27 percent and 33.98 percent homology respectively at CDS and amino acid levels has only the functions of regulating and controlling the spike number, tillering number and plant height (Dixonet al, 2018, plant cell; dixonet al, 2020, journ air experiment Botany). Therefore, the gene of the invention can be combined with an over-expression promoter in a plant and then introduced into a proper expression vector to transform a plant host, so that the tillering angle of the plant is changed, and the yield of the plant is regulated.
Description of the drawings:
FIG. 1 is a graph showing the relative expression level of TaTB1-2-A gene in the axillary buds of overexpressed transgenic wheat. Detecting the expression quantity of the TaTB1-2-A gene in axillary buds of transgenic wheat by adopting a real-time reverse transcription real-time quantitative PCR method, wherein the horizontal axis represents different transgenic wheat strains, 19# is a negative strain obtained in the genetic transformation process, and OE2 and OE5 are positive strains; the vertical axis represents: the ratio of the expression level of TaTB1-2-A of the transgenic strain relative to that of a wild-type field control plant, and the internal reference gene is Actin. ns indicates no significant difference, and x indicate very significant differences.
FIG. 2 is a diagram of the characterization of the trefoil phase over-expressed TaTB1-2-A gene transgenic wheat Fielder axillary bud phenotype (a) and a data statistical diagram (b). Wherein Bar in a is 1mm. The horizontal and vertical axes are shown in the figure 1 legend. ns indicates no significant difference, and x indicates very significant difference.
FIG. 3 is a graph of characterization of the field tillering phenotype of transgenic wheat over-expressing the TaTB1-2-A gene during the jointing and maturation phases and a graph of data statistics. a is the angle phenotype of the field tillering of the transgenic wheat of the jointing period over-expressed TaTB1-2-A gene, wherein Bar is 10cm; b is a phenotype statistical graph of different strains in a; c is a statistical chart of the field tiller number of the transgenic wheat with the mature period over-expressed TaTB1-2-A gene. The horizontal and vertical axes are shown in the figure 1 legend. ns represents no significant difference, x represents a significant difference, and x respectively represent very significant differences.
The specific embodiment is as follows:
the following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials, reagents and the like used in the examples are all commercially available unless otherwise specified.
The materials used were Fielder background transgenic materials TaTB1-2-A-OE2 and TaTB1-2-OE5 obtained by wheat variety Walker, fielder, agrobacterium-mediated genetic transformation, negative plants TaTB1-2-A-19#.
Example 1: verification of the regulatory action of the TaTB1-2-A Gene on tillering Using transgenes
cDNA is obtained by selecting the RNA reverse transcription of the axillary bud of the expected node, CDS of the TaTB1-2-A gene is amplified by a PCR method, and is constructed on a pWMB110 vector, and the promoter is Ubi of corn.
The primers used for PCR amplification of the CDS of the TaTB1-2-A gene were as follows:
| primer name | Primer sequence (5 'to 3') |
| TaTB1-1A-CDS-F | ATGGAAATGGAAATGGGGATG |
| TaTB1-1A-CDS-R | TCAGTACTGGATGCGTGGTT |
The method described in the "high color nepheline et al (2015) patent (patent number CN 201310726478.8) is specifically referred to using agrobacterium-mediated transgene technology. TaTB1-2-A was overexpressed in the wheat variety Fielder. T (T) 0 RNA extracted from the generation leaves is found by identifying the expression quantity of the TaTB1-2-A gene, the TaTB1-2-A-OE2 and the TaTB1-2-A-OE5 are transgenic positive plants, and negative plants TaTB1-2-A-19# are reserved as negative controls after the simultaneous tissue culture with the positive plants. T for obtaining transgenic plants by greenhouse planting 1 Analysis of the expression level of TaTB1-2-A in axillary bud tissues of transgenic positive plants, negative plants and receptor Fielder in the trefoil period by qRT-PCR analysis shows that the expression level of TaTB1-2-A in axillary buds of the two positive lines is obviously up-regulated, and the expression level of TaTB1-2-A in axillary buds of the negative lines is not obviously different from that in Fielder (figure 1).
The fluorescent quantitative PCR amplification primers for the TaTB1-2-A gene are as follows:
| primer name | Primer sequence (5 'to 3') |
| TaTB1-2AQ-F | CTCACCATCAACACCCTCCT |
| TaTB1-2AQ-R | CCGTGCATATCTTGCTGTGT |
| Actin-F | TTGCACCAAGCAGCATGAA |
| Actin-R | AACCACCGATCCAGACACTGTA |
To further determine candidate gene function, consider T 1 Axillary buds and tillering phenotype of the generation line. The three leaf stage TaTB1-2-A-OE2 and TaTB1-2-A-OE5 were found to be significantly shorter than the first axillary buds of Fielder and TaTB1-2-A-19# (FIG. 2), indicating that TaTB1-2-A also functions to regulate tillering by inhibiting axillary bud elongation, which is similar to the tillering inhibition function of TaTB1-1-D (Dixonet. 2018, plant cell). The tillering angles of TaTB1-2-A-OE2 and TaTB1-2-A-OE5 in the mature period are obviously increased compared with those of the Fielder and TaTB1-2-A-19# and the function of the TaTB1-like gene for regulating the tillering angle is not reported yet (figure 3). Thus, it was found that the tillering angle was significantly increased while the elongation of axillary buds was significantly inhibited by the overexpression of TaTB1-2-A in Fielder. The regulation and control effect of TaTB1-2-A on the tillering angle has important significance for increasing the planting density of crops, improving the photosynthetic efficiency of groups, increasing the ventilation and reducing the incidence of scab.
The extraction method of the plant tissue RNA and the identification method of the relative expression quantity are as follows:
1. extracting total RNA by using a Trizol kit method: the total RNA extraction step is strictly in accordance with TransZoL TM The specification is carried out as follows: (1) 1mL of TransZoL is added in advance TM Reagent into 2mL centrifuge tube; (2) Taking 0.15g young wheat ear, placing into a mortar cooled by liquid nitrogen in advance, rapidly grinding into powder, and grinding to obtain materialSoaking in liquid nitrogen all the time; (3) Transfer of the ground sample to a pre-applied TransZoL TM In a centrifuge tube of the reagent, 1ml of TransZoL is used per one TM Adding 0.2ml chloroform, shaking vigorously for 15s, standing at room temperature for 5min, and separating nucleic acid from protein completely; (4) Centrifuge in a refrigerated centrifuge at 4℃and 10000rpm for 15min. At this point the sample separated into three layers, the upper colorless aqueous phase, the middle layer and the lower pink organic phase. RNA is mainly in the water phase; (5) Carefully transfer the upper aqueous phase into a clean tube at 1mL of TransZoL TM Adding 0.5mL isopropyl alcohol standard into the extract, mixing, standing at room temperature for 10min; (6) Centrifuge at 12000rpm for 10min at 4℃in a refrigerated centrifuge. Discarding the supernatant, and forming colloidal precipitate in the test tube; (7) Discarding the supernatant, adding 75% ethanol prepared with 1mL DEPC treated water, and vigorously vortexing; (8) Centrifuging at 4deg.C and 1200rpm for 5min in a refrigerated centrifuge, and discarding supernatant; (9) Removing 75% ethanol, drying in a super clean bench for 5min, and dissolving with 50-100 μl RNA solution; (10) Incubating for 10min at 55-60 ℃, and preserving the sample at-80 ℃ for standby.
2. First strand cDNA sequence synthesis was performed using a reverse transcription kit: synthesis of first strand cDNA reference PrimeScript TM II 1st strand cDNA synthesis Kit description: (1) dNTP mix, oligo dT Primer and template RNA are added to a clean test tube without RNase; (2) Gently mixing, incubating at 65deg.C for 5min, rapidly cooling in ice box, and adding 5× PrimeScript II Buffer, RNase Inhibitor, primeScript II RTase and RNase free ddH2O; (3) gently mixing and incubating at 42℃for 60min. (4) stopping the reaction at 95 ℃ for 5min to inactivate the enzyme. The reverse transcription sample is stored in a refrigerator at-80 ℃ for standby.
3. Real-time fluorescent quantitative PCR (qRT-PCR) procedure: (1) The reverse transcription reaction system and amplification procedure of total RNA were described in HiScript III RT SuperMix for qPCR (Vazyme) kit to obtain cDNA of each sample. Based on fluorescent quantitative reagentsqPCR SYBR Green Master Mix the operation shows that the wheat Actin gene is used as an internal reference in real time in Roche 480The fluorescent quantitative PCR instrument is used for detecting the expression condition of the genes. One parallel experiment was run with 3 replicates, with at least three biological replicates per experiment.
(2) qRT-PCR reaction system:
(3) Amplification procedure:
experimental results Using 2 -△△CT And (3) carrying out data analysis by a method, namely calculating the relative expression quantity of the target gene relative to the untreated sample at different time points after treatment according to the obtained CT value.
Claims (2)
1. Wheat geneTaTB1-2-AApplication of the gene in enhancing the tillering angle of wheat, namely the gene from common wheat for water whiteTaTB1-2-AThe cDNA sequence of (C) is shown as SEQ ID NO: 1.
2. Comprising the gene as claimed in claim 1TaTB1-2-AThe expression cassette, the recombinant vector or the recombinant microorganism in enhancing the tillering angle of wheat.
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Non-Patent Citations (3)
| Title |
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| Genome-Wide Identification and Expression Profiling of the TCP Family Genes in Spike and Grain Development of Wheat (Triticum aestivum L.);Junmin Zhao等;Front. Plant Sci.;第9卷;1282 * |
| 小麦分蘖角度TaTAC1基因同源克隆及表达分析;曹鑫;邓梅;张正丽;刘宇娇;杨希兰;周红;刘亚西;;植物遗传资源学报(01);125-132 * |
| 控制四倍体小麦穗分枝性状基因WRS的定位与候选基因分析;王左君;中国优秀硕士学位论文全文数据库 农业科技辑(第01期);D047-257 * |
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