阅读说明：本技术 一种白三叶转录因子TrNAC及其编码序列和应用 (Trifolium repens transcription factor TrNAC and coding sequence and application thereof ) 是由 彭燕 蒲棋 吴星 章有知 程碧真 孙梦薇 贾彤 冯华昊 于 2020-07-21 设计创作，主要内容包括：本发明涉及生物技术领域,公开了一种白三叶转录因子TrNAC及其编码序列和应用。本发明所述白三叶转录因子TrNAC氨基酸序列如SEQ ID NO:1所示。本发明提供的白三叶转录因子TrNAC具有潜在的抗非生物胁迫能力,其编码序列在干旱、盐、低温、重金属胁四种非生物逆境胁迫下转录水平均有明显升高,将其编码序列转入到拟南芥中,转基因植株较野生型生长速度更快,叶片更大型,同时在自然干旱胁迫下也表现出明显的抗旱性,对培育高生长性能和抗非生物胁迫能力的植物具有重要意义。(The invention relates to the technical field of biology, and discloses a trefoil alba transcription factor TrNAC, a coding sequence and application thereof. The trefoil alba transcription factor TrNAC amino acid sequence is shown in SEQ ID NO 1. The transcription factor TrNAC of the white clover has potential abiotic stress resistance, the transcription water average of the coding sequence of the transcription factor TrNAC is obviously increased under the four abiotic stresses of drought, salt, low temperature and heavy metal coelioma, the coding sequence is transferred into arabidopsis thaliana, a transgenic plant has higher growth speed and larger leaves compared with a wild plant, and simultaneously, the coding sequence also shows obvious drought resistance under natural drought stress, and has important significance for cultivating plants with high growth performance and abiotic stress resistance.)

1. A Trifolium repens transcription factor TrNAC is characterized in that the amino acid sequence is shown in SEQ ID NO. 1.

2. The coding sequence of trefoil transcription factor TrNAC of claim 1.

3. The coding sequence of claim 2, wherein the nucleotide sequence is 1-882bp of the sequence shown in SEQ ID NO. 2 or the sequence shown in SEQ ID NO. 2.

4. A recombinant vector, recombinant bacterium, transgenic cell line or expression cassette comprising the coding sequence of claim 2 or 3.

5. Use of the trefoil alba transcription factor TrNAC according to claim 1 for improving stress resistance in plants or for improving growth performance in plants.

6. The use of claim 5, wherein the stress resistance is selected from one or more of drought resistance, salt resistance, cold resistance and heavy metal resistance.

7. The use of claim 5, wherein the plant is Trifolium repens or Arabidopsis thaliana.

8. A method for improving the stress resistance and/or growth performance of plants, which is characterized in that the coding sequence of claim 2 or 3 is introduced into target plants by using genetic engineering technology to improve the stress resistance and/or growth performance of the target plants.

9. The method of claim 8, wherein the genetic engineering technique is Agrobacterium-mediated inflorescence dip-dyeing.

Technical Field

The invention relates to the technical field of biology, in particular to a trefoil alba transcription factor TrNAC and a coding sequence and application thereof.

Background

Trifolium repens L is perennial cold season leguminous forage and is widely planted in southwest of China, middle and lower reaches of Yangtze river and other areas. The forage grass has the characteristics of high yield, good palatability, high nutrient content and digestibility, strong biological nitrogen fixation capacity and the like, and becomes the main leguminous forage grass cultivated and utilized all over the world. In addition, the trifolium repens stems and leaves are beautiful, have high regeneration speed, have the functions of preventing water and soil loss, adsorbing dust, purifying air and the like, and become main grass seeds for ornamental lawns and greenbelts in temperate regions. However, the white clover likes cold and humid climate, has shallow root system, low transpiration regulation capability and poor drought tolerance, is generally planted in shallow and barren soil, and has serious influence on growth, yield and disease resistance under water shortage condition. The productivity and durability of trifolium repens planted in drought-prone areas are limited. Therefore, drought is one of the main factors limiting the high quality and high yield of the white clover. Therefore, the development and the functional verification of genes related to the improvement of the growth and the drought resistance of the white clover lay an important foundation for the improvement of the yield and the stress resistance of the white clover.

Disclosure of Invention

In view of the above, the present invention aims to provide a trefoil alba transcription factor TrNAC and its coding sequence, so that the TrNAC exhibits higher expression level under drought, salt, cold and heavy metal stress, and at the same time, can improve plant growth performance, and can be used as a potential plant stress-resistant protein and a growth performance-improving protein;

another purpose of the present invention is to provide the application of TrNAC described above in improving stress resistance of plants;

it is a further object of the present invention to provide the use of TrNAC described above for enhancing plant growth performance.

In order to achieve the above purpose, the invention provides the following technical scheme:

a Trifolium repens transcription factor TrNAC has an amino acid sequence shown in SEQ ID NO 1. The invention takes a trilobate variety 'Ladinol' as a material, obtains a transcription factor TrNAC which can show higher potential in abiotic stress from a gene sequence, verifies an expression mode of a coding sequence of the transcription factor TrNAC in drought, salt, low temperature and heavy metal stress through qRT-PCR, and shows that the transcription water average of the coding sequence is obviously increased under the four abiotic adversity stresses.

Under abiotic stress, the expression level of the coding sequence of the transcription factor TrNAC in roots and leaves is remarkably changed, and the expression level is different under each stress condition and time point. The coding sequence is transferred into arabidopsis thaliana by means of gene engineering, and a transgenic plant has higher growth speed, larger leaves and longer and more root systems compared with a wild type plant; also shows obvious drought resistance under natural drought stress.

Meanwhile, the invention also provides a coding sequence of the transcription factor TrNAC, and in a specific embodiment of the invention, the nucleotide sequence of the coding sequence is shown as 1-882bp in the sequence shown in SEQ ID NO. 2 or as shown in SEQ ID NO. 2. In addition, the invention also provides a technical scheme of a recombinant vector, a recombinant bacterium, a transgenic cell line or an expression cassette containing the coding sequence, so that a genetic engineering means is used for expressing the transcription factor TrNAC.

Based on the effects of the transcription factor TrNAC in abiotic stress of plants and growth promotion (high growth speed, larger leaves, longer root systems and more), the invention provides the application of the transcription factor TrNAC in improving the stress resistance of plants or the growth performance of the plants.

In a specific embodiment of the invention, the stress resistance is selected from one or more of drought resistance, salt resistance, cold resistance and heavy metal resistance; the plant is Trifolium repens or Arabidopsis thaliana.

According to the application, the invention also provides a method for improving the stress resistance and/or the growth performance of the plant, and the coding sequence is introduced into a target plant by utilizing a genetic engineering technology to improve the stress resistance and/or the growth performance of the target plant.

Wherein, the genetic engineering technology can adopt the technical means of gene introduction already recorded in the field, such as an agrobacterium-mediated inflorescence dip dyeing method, recombinant plasmids, recombinant bacteria, transgenic cell lines or expression cassettes and the like to realize over-expression of the introduced genes; in a specific embodiment of the invention, the genetic engineering technology is an agrobacterium-mediated inflorescence dip dyeing method.

According to the technical scheme, the transcription factor TrNAC has potential abiotic stress resistance, the transcription water average of the coding sequence of the transcription factor TrNAC is obviously increased under four abiotic stresses of drought, salt, low temperature and heavy metal, the coding sequence is transferred into Arabidopsis, a transgenic plant has higher growth speed and larger leaves compared with a wild plant, and simultaneously, the coding sequence also shows obvious drought resistance under natural drought stress, and the coding sequence has important significance for cultivating plants with high growth performance and abiotic stress resistance.

Drawings

FIG. 1 shows the quantitative expression analysis of Trifolium pratense TrNAC under four abiotic stresses; wherein the ordinate is relative expression, the abscissa is treatment time (0, 1.5, 3, 6, 12, 24h), the left side in each column is relative expression of the root, and the right side is relative expression of the leaf;

FIG. 2 shows the growth of wild type and over-expressing TrNAC Arabidopsis plants under normal conditions; from left to right are WT (wild type), overexpression lines OE1, OE1, OE8 and OE12, respectively;

FIG. 3 shows the growth of wild type and over-expressed TrNAC Arabidopsis plants under drought stress; wherein, the upper left and the lower right are wild plants, and the upper right and the lower left are over-expression plants.

Detailed Description

The invention discloses a trefoil alba transcription factor TrNAC and a coding sequence and application thereof, and can be realized by appropriately improving process parameters by taking the contents of the text as reference by a person skilled in the art. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the trefoil alba transcription factor TrNAC and its coding sequence and use of the present invention have been described by way of example, it will be apparent to those skilled in the art that the techniques of the present invention may be implemented and used by modifying or appropriately modifying and combining the trefoil alba transcription factor TrNAC and its coding sequence and use without departing from the spirit, scope and spirit of the invention.

The open reading frame of the coding sequence of the trefoil Baicalensis transcription factor TrNAC is 882bp, and 293 amino acids are coded.

The coding sequence of the Trifolium repens transcription factor TrNAC predicts that the molecular formula of the Trifolium repens transcription factor TrNAC is C through a ProParam tool₁₅₃₁H₂₃₀₁N₄₀₇O₄₅₇S₁₁The protein is a stable non-transmembrane hydrophilic protein and has a plurality of phosphorylation sites, and is a protein which is predicted to be composed of 22.87% of α helix, 9.90% of β turn, 19.11% of extension chain and 48.12% of irregular coil through the secondary structure and the tertiary structure.

In the comparative experiments of the present invention, unless otherwise specified, all groups were kept identical except for the artificially set differences (e.g., differences in the presence or absence of the coding sequence for the trefoil transcription factor TrNAC of the present invention).

The trefoil alba transcription factor TrNAC provided by the invention, the coding sequence and the application are further explained below.