AhPILS gene for regulating plant lateral branch development and promoter and application of AhPILS gene

Abstract

The invention discloses an AhPILS gene for regulating plant lateral branch development and a promoter and application thereof, and belongs to the technical field of agricultural biology. The invention provides an AhPILS gene for regulating plant lateral branch development. The nucleotide sequence of the AhPILS gene is as shown in SEQ ID NO. 1. The amino acid sequence of the AhPILS protein coded by the AhPILS gene is as shown in SEQ ID NO. 2. The nucleotide sequence of the promoter of the AhPILS gene is as shown in SEQ ID NO. 3; and G / A base mutation exists at the-895bp position of the sequence as shown in SEQ ID NO.3. The gene AhPILS for controlling peanut lateral branch development is positioned through a MutMap technology, a foundation is laid for application of the gene AhPILS in control of peanut lateral branch development, and the gene has important significance in peanut plant type improvement.

Description

Technical Field

[0001] The present invention relates to the field of agricultural biotechnology, and particularly relates to an AhPILS gene that regulates plant lateral branch development, its promoter, and applications thereof. Background Art

[0002] Peanut is an important oil crop and cash crop. The peanut lateral branch angle (or called branching angle) is an important agronomic trait that determines the peanut plant type, directly affecting peg penetration into the soil, planting density, cultivation management, mechanized harvesting, and Aspergillus flavus control. Currently, the main peanut varieties promoted in China are mainly upright types, while the peanuts in the United States are mainly creeping varieties. Compared with the upright type, the creeping peanut has a low planting density, less seed consumption, and does not require chemical control. During harvesting, the creeping peanut is more suitable for two-stage mechanized harvesting. When harvesting, the peanuts are first dug up and inverted so that the pods face upwards. Due to the support of the lateral branches, direct contact between the pods and the soil is avoided, facilitating in-situ drying of the peanuts and effectively reducing Aspergillus flavus contamination. The inventors' previous research showed that picking the fruits 3 - 4 days after the peanuts emerge and are dried in the sun can not only increase the percentage of plump fruits and plump kernels, but also achieve yield increase. Therefore, creeping and semi-creeping peanuts will be one of the important directions for peanut breeding in China.

[0003] Currently, most of the creeping and semi-creeping peanut germplasms in China are derived from foreign introductions or wild resources, and many traits are quite different from those of the main domestic varieties. It is difficult to break the linkage between unfavorable traits and plant type using conventional breeding methods. In addition, the lateral branch angle is sensitive to environmental factors, which also affects the accuracy of field screening. Therefore, precisely mapping the key genes controlling the lateral branch angle and using molecular breeding methods to improve the peanut plant type and cultivate new creeping and semi-creeping peanut varieties are important directions to support the high-quality development of the peanut industry. Aiming at the above deficiencies, the present invention has identified a key gene and its promoter involved in regulating peanut lateral branch development, and it is of great significance to apply this gene to the improvement of peanut plant type. Summary of the Invention

[0004] The object of the present invention is to provide an AhPILS gene that regulates plant lateral branch development, its promoter, and applications thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention lays a foundation for the application of the AhPILS gene in controlling peanut lateral branch development, and it is of great significance to apply this gene to the improvement of peanut plant type.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an AhPILS gene that regulates plant lateral branch development, and the nucleotide sequence of the AhPILS gene is as shown in SEQ ID NO.1.

[0007] The present invention also provides an AhPILS protein for regulating the development of plant lateral branches, and the amino acid sequence of the AhPILS protein is shown in SEQ ID NO.2.

[0008] The present invention also provides the application of the AhPILS gene or the AhPILS protein in regulating the development of plant lateral branches.

[0009] Optionally, the plant includes peanut.

[0010] The present invention also provides the promoter of the AhPILS gene, and the nucleotide sequence of the promoter is shown in SEQ ID NO.3;

[0011] There is a G / A base mutation at -895bp of the sequence shown in SEQ ID NO.3.

[0012] The present invention also provides the application of the promoter in regulating the plant plant type.

[0013] Optionally, if the base at -895bp of the sequence shown in SEQ ID NO.3 is A, the plant plant type is erect type.

[0014] Optionally, the plant includes peanut.

[0015] The present invention also provides a method for judging the plant plant type, including the steps of detecting the promoter sequence of the AhPILS gene of the plant to be tested and making a judgment according to the detection result;

[0016] The nucleotide sequence of the AhPILS gene is shown in SEQ ID NO.1;

[0017] The nucleotide sequence of the promoter is shown in SEQ ID NO.3.

[0018] Optionally, if the base at -895bp of the sequence shown in SEQ ID NO.3 is A, it is judged that the plant to be tested is of erect type;

[0019] The plant includes peanut.

[0020] The present invention discloses the following technical effects:

[0021] The present invention uses the prostrate peanut variety Georgia-06G (G06G) 60 An erect mutant eg06g obtained by Coγ-ray radiation mutagenesis. Quantitative analysis of various hormones and their derivatives on the proximal and distal sides of the lateral branch base of G06G and eg06g found that the distribution of auxin at the lateral branch base of the eg06g mutant changed, and the auxin on the proximal side of the lateral branch base of eg06g was higher than that on the distal side, promoting cell elongation and causing the mutant eg06g to transform into an erect type.

[0022] The gene AhPILS that controls peanut lateral branch development was located by the MutMap technique. Through the analysis of SNP mutation sites in the candidate interval, it was found that there was a mutation from G to A at the -895 position of the AhPILS promoter (SEQ ID NO.3). After the mutation, a CAAT element was added to this region. Through sequencing verification, when the base at this site in the plant is A, the plant type of this plant is erect. The present invention lays a foundation for the application of the AhPILS gene in controlling peanut lateral branch development, and it is of great significance to apply this gene to peanut plant type improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 For the phenotypes of G06G erect type and mutant eg06g; A: Seedlings of G06G and eg06g at 20 days; B: Plants of G06G and eg06g at the harvest stage; C: Plant height at the harvest stage; D: Lateral branch length at the harvest stage; E: Number of branches at the harvest stage;

[0025] Figure 2 For the mapping result of mutant eg06g and the mutation site of candidate gene AhPILS;

[0026] Figure 3 For the prediction of cis-acting elements in the promoter region; A: Prediction of cis-acting elements in the original sequence; B: Prediction of cis-acting elements in the mutant sequence;

[0027] Figure 4 For the detection of mutant bases in erect and prostrate peanuts; A: Comparing the bases in erect peanuts using the Peanutbase website; B: Detection of bases in the prostrate variety of the PH population; C: Detection of bases in the erect variety of the PH population; D: Detection of bases in the prostrate germplasm resources; E: Detection of bases in the erect germplasm resources;

[0028] Figure 5 For the contents of the main and minor components of auxin; A: Indole-3-acetic acid (IAA); B: Indoleacetic acid-aspartic acid (IAA-Asp); C: Indoleacetic acid-glutamic acid (IAA-Glu); D: Indole-3-carbaldehyde (ICAId); E: Indole-3-carboxylic acid (ICA); F: L-tryptophan (TRP). DETAILED DESCRIPTION OF THE EMBODIMENTS

[0029] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and implementation schemes of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] The present invention utilizes the prostrate peanut variety Georgia-06G (G06G) 60 An erect mutant eg06g obtained by Coγ-ray radiation mutagenesis was used to map a gene AhPILS (M37AJ6) controlling the peanut lateral branch angle through the MutMap technique. This gene encodes a member of the PIN-formed (PIN) and structurally similar PIN-like (PILS) families and is mainly responsible for auxin transport. Specifically as follows.

[0036] I. Obtaining and phenotypic analysis of the mutant

[0037] The present invention starts from 60An erect peanut mutant eg06g was identified from a mutant library of the prostrate peanut cultivar Georgia-06G (G06G) irradiated with Co-γ rays. Its lateral branch angle was significantly smaller than that of G06G. The lateral branch angle of the prostrate peanut G06G was 83.7°, while that of the erect mutant eg06g was 36.4°. At harvest, the main stem height, lateral branch length, and number of lateral branches of G06G and eg06g were counted. The average main stem height of G06G was 17.2 cm, the length of the first lateral branch was 47.2 cm, and the average number of lateral branches was 17. Compared with G06G, the main stem height of eg06g was 129.6% higher, with an average main stem height of 39.5 cm; the length of the first lateral branch was 13.1% longer, with the first lateral branch length being 53.4 cm; and the number of branches was 29.4% less, with an average number of lateral branches being 12( Figure 1 ).

[0038] II. Construction of a segregating population and BSA-seq mapping of candidate genes

[0039] The prostrate cultivar G06G was used as the female parent and the erect mutant eg06g as the male parent for hybridization to construct the hybrid PH6 population. MutMap was used for gene mapping and functional analysis of this erect mutant.

[0040] According to the F2 phenotype survey results, 30 prostrate and 30 erect lines with extreme phenotypes were selected for pooling, and whole-genome resequencing was performed together with the parents. Based on the resequencing results, the QTL controlling the lateral branch angle of eg06g was initially mapped to the interval of Chr19: 120 - 139 Mb by SNP-index association analysis of variant sites( Figure 2 ). Combining the transcriptome sequencing results of the materials on the near-ground side and the dorsal side at the base of the first lateral branch of G06G and eg06g, the differentially expressed genes in this interval between G06G and eg06g were analyzed, and a gene that might affect the lateral branch angle was predicted. This gene encodes a PIN-like (PILS) protein and is involved in auxin transport. It was named AhPILS( Figure 2 ). The nucleotide sequence of the AhPILS gene is shown in SEQ ID NO.1, and the amino acid sequence of the encoded AhPILS protein is shown in SEQ ID NO.2.

[0041] Analysis of SNP variant sites within the candidate interval found that there was a G-to-A mutation (shaded position) at the -895 position of the AhPILS promoter (SEQ ID NO.3), and this mutation added a CAAT element to this region( Figure 3 ).

[0042] SEQ ID NO.1 (Nucleotide sequence of the AhPILS gene):

[0043]

[0044] SEQ ID NO. 2 (Amino acid sequence of the protein encoded by the AhPILS gene):

[0045] MQFWKLFITALMPVLKVLLITALGAFLALDRFNVLRDTARKHLNTLVYYVFTPALVCSILTKTITFKNLVMLWFMPLNILLTFIVGTALGWLFMKITKAPNHMYGLVLGCCAAGNLGNLPLIIMPTVCKESNSPFGAVDLCYKKGMGYASLSMAIGNIYIWTFVYNIVRLYSSNKTLDNNSTLEKDSVKNSTEDLSETVDDHENQLQIETTFSHERAKLPKLAKIVKTLVEKLNLKVLLAPATVGSIVGLVMGGITPFRKLFVGDDAPLRVLEDSTSMVGDAAIPAVTLLVGANLLKGLKGSGMKVPLLLGILVVRYIALPMLGVCIVKGAIHFGLINHDPLYQFMLLLQYALPPAISISTITQLFGAGETECSIIMLATYACAAVSLTLWSTFFMWLVL*;

[0046] SEQ ID NO. 3 (Nucleotide sequence of the AhPILS gene promoter):

[0047]

[0048] Note: The shadow - marked "R" is the G / A mutation site.

[0049] III. Analysis of Mutation Sites in Different Peanut Plant Types

[0050] By comparing through the Peanutbase website, it was found that in the erect peanut varieties "Fuhuasheng" and "Shitoushanqi", the base at the - 895 site in the promoter region of the AhPILS gene is A, which is the same as the base at this site in the erect mutant eg06g ( Figure 4 in A).

[0051] Base sequencing was performed on the - 895bp site in the promoter region of the AhPILS gene of prostrate and erect plants in the PH6 population. It was found that in the PH6 population, the base at this site in prostrate plants is the same as that in the prostrate parent G06G, and all bases are G ( Figure 4 in B); in the PH6 population, the base at this site in erect plants is the same as that in the erect parent eg06g, and all bases are A ( Figure 4 in C).

[0052] Base sequencing was performed on the - 895bp site in the promoter region of the AhPILS gene of erect peanut germplasm resources and prostrate peanut germplasm resources. The results showed that all bases at this site in prostrate peanut germplasm resources are G ( Figure 4 in D), and part of the bases at this site in erect peanut germplasm resources are A and part are G ( Figure 4 in E). The above results indicate that in peanuts, if the base at this site is G, the plant type of peanuts is likely to be prostrate, and if the base at this site is A, the plant type of peanuts must be erect.

[0053] IV. Quantitative Analysis of Hormones and Their Derivatives

[0054] To reveal the role of auxin in the formation of the lateral - branch angle of eg06g, quantitative analysis of various hormones and their derivatives on the adaxial and abaxial sides of the lateral - branch base of G06G and eg06g was carried out.

[0055] The content of indole - 3 - acetic acid (IAA) in the erect mutant eg06g changed significantly compared with that in G06G. In G06G, the content of indole - 3 - acetic acid on the adaxial side of the lateral - branch base was lower than that on the abaxial side, while in the erect mutant eg06g, the content of indole - 3 - acetic acid on the adaxial side was higher than that on the abaxial side ( Figure 5 in A). Indole - 3 - acetic acid - aspartic acid (IAA - Asp) and indole - 3 - acetic acid - glutamic acid (IAA - Glu) are the main storage forms of auxin in plants and can be converted into IAA by the ILR1 amidohydrolase. Their contents on the adaxial and abaxial sides in the erect mutant eg06g are significantly higher than those in G06G ( Figure 5In B-C). Indole-3-carboxaldehyde (ICAId) has a growth-promoting effect, and its content on the proximal side of eg06g is significantly higher than that of other samples ( Figure 5 In D). Some other auxin-related substances ( Figure 5 In E-F), there are also significant differences in their contents between G06G and eg06g. These results indicate that the distribution of auxin at the base of the lateral branches of the eg06g mutant has changed. The auxin on the proximal side of the base of the lateral branches of eg06g is higher than that on the dorsal side, promoting cell elongation and causing the mutant eg06g to transform into an upright type.

[0056] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. An AhPILS gene for regulating plant lateral branch development, characterized in that, The nucleotide sequence of the AhPILS gene is shown as SEQ ID NO.

1.

2. An AhPILS protein for regulating plant lateral branch development, characterized in that, The amino acid sequence of the AhPILS protein is shown as SEQ ID NO.

2.

3. Use of the AhPILS gene according to claim 1 or the AhPILS protein according to claim 2 in regulating the development of plant lateral branches.

4. The application according to claim 3, wherein The plant includes peanut.

5. The promoter of the AhPILS gene according to claim 1, characterized in that, The nucleotide sequence of the promoter is shown as SEQ ID NO.3; There is a G / A base mutation at -895bp of the sequence shown in SEQ ID NO.

3.

6. Use of the promoter according to claim 5 in regulating plant plant type.

7. The application according to claim 6, characterized in that, If the base at -895bp of the sequence shown in SEQ ID NO.3 is A, the plant plant type is erect type.

8. The application according to claim 6, wherein The plant includes peanut.

9. A method for judging the plant type, characterized in that, It includes the steps of detecting the promoter sequence of the AhPILS gene of the plant to be tested and making a judgment according to the detection result; The nucleotide sequence of the AhPILS gene is shown as SEQ ID NO.1; The nucleotide sequence of the promoter is shown as SEQ ID NO.

3.

10. The method according to claim 9, characterized in that, If the base at -895bp of the sequence shown in SEQ ID NO.3 is A, it is judged that the plant to be tested is of erect type; The plant includes peanut.

Images