WO2024244193A1 - Tatb1-2-a gene for controlling tillering angle of common wheat, and protein encoded thereby - Google Patents

Abstract

Provided are a gene TaTB1-2-A for controlling the tillering angle of common wheat, and a protein encoded thereby. A Wangshuibai TaTB1-2-A gene cDNA sequence is as shown in SEQ ID NO: 1, and a TaTB1-2-A gene encoding amino acid sequence is as shown in SEQ ID NO: 3. High expression of the TaTB1-2-A gene in wheat has the effect of significantly increasing the tillering angle, and the gene can be combined with an overexpression promoter in a plant and then introduced into a suitable expression vector and converted into a plant host, so as to change the tillering angle of the plant, and thus regulate and control the yield of the plant.

Description

A tillering angle control gene TaTB1-2-A in common wheat and its encoded protein 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 gene.

Background Art

Wheat (Triticum aestivum L.) is the largest crop planted in the world, which can feed more than 35% of the world's total population and provide various proteins, minerals, vitamins and other substances for humans. About 20% of the total calories consumed by humans are provided by wheat. my country is the world's largest wheat producer and consumer, and its wheat production accounts for about 17% of the world's total production. With the continuous increase in population, it is estimated that food 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 my country's food security and social stability.

Since the 1970s, the use of the semi-dwarf gene SD1 in rice and the Rht1 gene in wheat has almost doubled the world's grain production, basically solving the world's food security problem. This green revolution has become a classic case of using plant type to increase crop yields. In recent years, wheat high-yield breeding has encountered new bottlenecks. As wheat plant height decreases, it will bring some negative effects, such as dense overlapping of leaf layers leading to reduced light energy utilization. Wheat tillering is one of the important traits of plant type. A reasonable tillering angle can not only increase the sowing density, but also improve the photosynthetic efficiency of the group and the resistance of the plant, thereby affecting the yield and quality of wheat. Our province is located in the transition zone between the north and the south, with a changeable climate, especially in the Huainan area, where the climate is warm and humid, and there is more rain in the middle and late stages of wheat growth, resulting in frequent and repeated occurrences of fusarium head blight. The sowing density directly affects the ventilation and light transmission of the wheat field, so a reasonable plant type structure can reduce the harm caused by fusarium head blight. Therefore, exploring the genes that regulate wheat tillering angle and the mechanism of wheat plant architecture regulation can not only solve basic problems in plant science, but also have important practical significance for the breeding of new high-yield and high-quality wheat varieties.

Tillering angle refers to the angle between the lateral tiller and the main stem, and is an important component of plant type. According to the size of the tillering angle, plants can be divided into three types: clustered type (plant tillering angle is less than 20°), compact type (plant tillering angle is between 21-32°) and loose type (plant tillering angle is greater than 33°). Tillering angle is a complex agronomic trait that is closely related to the yield of crop populations and is regulated by genetics, hormones and the environment. In the study of crop type such as wheat and rice, the study of tillering angle has always been a hot topic and a difficult point. Therefore, the positioning, cloning and molecular mechanism analysis of genes that control tillering angle have important theoretical significance and application value for improving crop type and thus increasing yield.

Summary of the invention

The purpose of the present invention is to provide a gene TaTB1-2-A related to wheat tillering angle in view of the above-mentioned deficiencies in the prior art.

Another object of the present invention is to provide application of the gene.

The purpose of the present invention can be achieved through the following technical solutions:

A gene TaTB1-2-A from Wangshuibai that is related to wheat tillering growth. The cDNA nucleotide sequence of the TaTB1-2-A gene from Wangshuibai is shown as SEQ ID NO: 1.

The CDS of the gene TaTB1-2-A is shown in SEQ ID NO: 2.

The amino acid sequence of the gene TaTB1-2-A is shown in SEQ ID NO: 3.

The TaTB1-2-A can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

The above-defined DNA sequences 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 with proteins having the same functions.

Genes encoding the above-mentioned similar domain proteins in other plants also fall within the protection scope of the present invention.

The gene TaTB1-2-A related to wheat tillering angle, and the expression box, recombinant vector or recombinant microorganism containing the gene TaTB1-2-A derived from the gene TaTB1-2-A.

The application of the gene TaTB1-2-A is as follows (A1) or (A2): (A1) regulating the tillering angle of the plant; (A2) increasing or decreasing the tillering angle of the plant.

The plant is a monocot or a dicot; the monocot may be a grass plant.

The use of the expression cassette, recombinant vector or recombinant microorganism is characterized by the following (B1) or (B2): (B1) a transgenic plant with a changed tillering angle; (B2) a transgenic plant with an increased or decreased tillering angle.

The plant is a monocot or a dicot; the monocot may be a grass plant.

Beneficial effects:

The high expression of the TaTB1-2-A gene of the present invention has the effect of significantly increasing the tillering angle in wheat, while TaTB1-1-D, which has 42.27% and 33.98% homology with it at the CDS and amino acid levels, respectively, only has the function of regulating the number of spikelets, the number of tillers and the height of the plant (Dixon et al., 2018, Plant Cell; Dixon et al., 2020, Journal of Experimental Botany). Therefore, the gene of the present invention can be combined with the overexpression promoter in the plant and then introduced into a suitable expression vector and transformed into a plant host, thereby changing the tillering angle of the plant and regulating the plant yield.

Description of the drawings:

Figure 1 is a graph showing the relative expression of the TaTB1-2-A gene in the axillary buds of overexpressed transgenic wheat Fielder. The expression of the TaTB1-2-A gene in the axillary buds of transgenic wheat was detected by real-time reverse transcription and real-time quantitative PCR. The horizontal axis represents different transgenic wheat lines, of which 19# is the negative line obtained during the genetic transformation process, and OE2 and OE5 are positive lines; the vertical axis represents the ratio of the TaTB1-2-A expression of the transgenic lines relative to the wild-type Fielder control plants, and the internal reference gene is Actin. ns indicates no significant difference, and ** and *** indicate extremely significant differences.

Figure 2 is a diagram (a) and a statistical diagram (b) of the Fielder axillary bud phenotype identification of transgenic wheat overexpressing the TaTB1-2-A gene at the three-leaf stage. The bar in a is 1 mm. The horizontal and vertical axes are as shown in the legend of Figure 1. ns indicates no significant difference, and ** indicates an extremely significant difference.

Figure 3 is a phenotype identification diagram and data statistical diagram of Fielder tillering of transgenic wheat with overexpression of TaTB1-2-A gene at jointing stage and maturity stage. a is the Fielder tillering angle phenotype of transgenic wheat with overexpression of TaTB1-2-A gene at jointing stage, where Bar is 10cm; b is the phenotype statistical diagram of different strains in a; c is the Fielder tillering number statistical diagram of transgenic wheat with overexpression of TaTB1-2-A gene at maturity stage. The horizontal and vertical axes are as shown in the legend of Figure 1. ns indicates no significant difference, * indicates significant difference, and ** and *** both indicate extremely significant differences.

Specific implementation method:

The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials, reagents, etc. used in the examples are commercially available unless otherwise specified.

The materials used were wheat varieties Wangshuibai and Fielder, transgenic materials TaTB1-2-A-OE2 and TaTB1-2-OE5 with Fielder background obtained by Agrobacterium-mediated genetic transformation, and negative plant TaTB1-2-A-19#.

Example 1: Using transgenic technology to verify the regulatory effect of TaTB1-2-A gene on tillering

The cDNA was obtained by reverse transcription of RNA from water-white axillary buds of expected jointing, and the CDS of TaTB1-2-A gene was amplified by PCR and constructed into pWMB110 vector, whose promoter was Ubi of maize.

The primers used for PCR amplification of TaTB1-2-A gene CDS are as follows:

Using Agrobacterium-mediated transgenic technology, specifically refer to the method described in the patent of Gao Caixia et al. (2015) (patent number, CN201310726478.8). TaTB1-2-A was overexpressed in the wheat variety Fielder. RNA extracted from _T0 leaves was identified by identifying the expression of TaTB1-2-A gene. It was found that TaTB1-2-A-OE2 and TaTB1-2-A-OE5 were transgenic positive plants, and the negative plant TaTB1-2-A-19# was retained as a negative control after simultaneous tissue culture with the positive plants. The _T1 generation strains of transgenic plants were obtained by greenhouse cultivation. qRT-PCR analysis of the expression of TaTB1-2-A in the axillary bud tissues of transgenic positive plants, negative plants and recipient Fielder at the three-leaf stage showed that TaTB1-2-A was significantly upregulated in both positive strains, and the expression of TaTB1-2-A in the axillary buds of the negative strains was not significantly different from that of Fielder (Figure 1).

The primers used for quantitative PCR amplification of TaTB1-2-A gene are as follows:

In order to further determine the function of the candidate gene, the axillary bud and tiller phenotypes of the _T1 generation strains were examined. It was found that the length of the first axillary bud of TaTB1-2-A-OE2 and TaTB1-2-A-OE5 at the three-leaf stage was significantly shorter than that of Fielder and TaTB1-2-A-19# (Figure 2), indicating that TaTB1-2-A also plays a role in regulating the number of tillers by inhibiting the elongation of axillary buds, which is similar to the function of TaTB1-1-D in inhibiting tillering (Dixon et al., 2018, Plant Cell). At the mature stage, the tiller angle of TaTB1-2-A-OE2 and TaTB1-2-A-OE5 was significantly increased compared with Fielder and TaTB1-2-A-19#, and the function of TaTB1-like genes in regulating tiller angle has not been reported (Figure 3). It can be seen that overexpression of TaTB1-2-A in Fielder significantly inhibited the elongation of axillary buds and affected the number of tillers, while the tiller angle increased significantly. The regulatory effect of TaTB1-2-A on the tiller angle is of great significance for increasing crop planting density, improving the photosynthetic efficiency of the population, and increasing air permeability to reduce the incidence of fusarium head blight.

The extraction of RNA from the above plant tissues and the identification of relative expression levels are as follows:

1. Extraction of total RNA using Trizol kit: The steps of total RNA extraction were strictly carried out in accordance with the TransZoL ^TM instructions, as follows: (1) Add 1 mL of TransZoL ^TM reagent to a 2 mL centrifuge tube; (2) Take 0.15 g of wheat spikelets and place them in a mortar cooled with liquid nitrogen, and quickly grind them into powder. During the grinding process, the material was always immersed in liquid nitrogen; (3) Transfer the ground sample to a centrifuge tube pre-added with TransZoL ^TM reagent, add 0.2 ml of chloroform for every 1 ml of TransZoL ^TM , shake vigorously for 15 seconds, and let stand at room temperature for 5 minutes to completely separate nucleic acids from proteins; (4) Centrifuge in a refrigerated centrifuge at 4°C, 10,000 rpm for 15 minutes. At this time, the sample is divided into three layers, the upper colorless aqueous phase, the middle layer and the lower pink organic phase. RNA is mainly in the aqueous phase; (5) Carefully transfer the upper aqueous phase to a clean test tube, add isopropanol at a standard of 0.5 mL of isopropanol per 1 mL of TransZoL ^TM extract, invert to mix, and let stand at room temperature for 10 minutes; (6) Centrifuge at 4°C, 12000 rpm for 10 minutes in a refrigerated centrifuge. Discard the supernatant, and a gelatinous precipitate will form in the test tube; (7) Discard the supernatant, add 75% ethanol prepared with 1 mL of DEPC-treated water, and vortex vigorously; (8) Centrifuge at 4°C, 1200 rpm for 5 minutes in a refrigerated centrifuge, and discard the supernatant; (9) Remove all 75% ethanol, dry in a clean bench for 5 minutes, and add 50-100 μL of RNA dissolution solution to dissolve; (10) Incubate at 55°C-60°C for 10 minutes, and store the sample at -80°C for later use.

2. Synthesis of the first strand cDNA sequence using a reverse transcription kit: Synthesis of the first strand cDNA was performed according to the instructions of the PrimeScript ^TM Ⅱ 1st strand cDNA synthesis Kit: (1) Add dNTP Mixture, Oligo dT Primer and template RNA to a clean tube free of RNase; (2) Mix gently, incubate at 65℃ for 5 min, quickly cool in an ice box and add 5× PrimeScript II Buffer, RNase Inhibitor, PrimeScript II RTase and RNase free ddH ₂ O; (3) Mix gently, incubate at 42℃ for 60 min; (4) Terminate the reaction at 95℃ for 5 min to inactivate the enzyme. The reverse transcribed sample was stored in a -80℃ refrigerator for later use.

3. Real-time fluorescence quantitative PCR (qRT-PCR) procedure: (1) The total RNA reverse transcription reaction system and amplification procedure refer to the instructions of the HiScript III RT SuperMix for qPCR (Vazyme) kit to obtain cDNA of each sample. qPCR SYBR Green Master Mix operating instructions, wheat Actin gene as an internal reference, on the Roche 480 real-time fluorescence quantitative PCR instrument to detect gene expression. A parallel experiment was repeated 3 times, and each experiment had at least three biological replicates.

(2) qRT-PCR reaction system:

(3) Amplification procedure:

The experimental results were analyzed using the 2- ^△△CT method, that is, the relative expression of the target gene at different time points after treatment relative to the untreated sample was calculated based on the obtained CT value.

Claims (8)

A gene TaTB1-2-A related to the tillering angle of wheat, characterized in that the cDNA sequence of the Wangshuibai gene TaTB1-2-A from common wheat is as shown in SEQ ID NO: 1. The gene TaTB1-2-A related to wheat tillering angle according to claim 1 is characterized in that the CDS from the gene TaTB1-2-A is as shown in SEQ ID NO: 2. The gene TaTB1-2-A related to wheat tillering angle according to claim 1 is characterized in that the amino acid sequence from the gene TaTB1-2-A is as shown in SEQ ID NO: 3. An expression cassette, recombinant vector or recombinant microorganism containing the gene TaTB1-2-A according to claim 1 or 2. The application of the gene TaTB1-2-A described in claim 1 or 2 is characterized by the following (A1) or (A2): (A1) regulating the tillering angle of the plant; (A2) enhancing or reducing the tillering angle of the plant. The use as claimed in claim 5 is characterized in that: the plant is a monocotyledon or a dicotyledon; the monocotyledon can be a grass plant. The use of the expression cassette, recombinant vector or recombinant microorganism according to claim 4 is characterized by being as follows (B1) or (B2): (B1) a transgenic plant cultivated with a changed tillering angle; (B2) a transgenic plant cultivated with an increased or decreased tillering angle. The use as claimed in claim 7 is characterized in that: the plant is a monocotyledon or a dicotyledon; the monocotyledon can be a grass plant.