CN-122012593-A - Application of LcCBP b-LcALY2 molecular module in regulation and control of growth and development of leymus chinensis and setting rate

CN122012593ACN 122012593 ACN122012593 ACN 122012593ACN-122012593-A

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses application of LcCBP b-LcALY2 molecular modules in regulating and controlling growth and development of leymus chinensis and setting percentage. The invention discovers that the calmodulin binding protein encoding gene LcCBP b negatively regulates the growth, development and fruiting rate of the leymus chinensis for the first time, and the yeast double-hybrid experiment discovers that LcCBP b interacts with LcALY protein to regulate the related characters of the fruiting rate of the leymus chinensis. LcCBP60B negatively regulates LcALY2 protein activity and inhibits the regulation of LcALY-encoded THO protein complex 4B in the flowering and spike formation processes of leymus chinensis. The LcCBP b-LcALY2 molecular module verified by the invention provides a new view for deeply understanding the regulation mechanism of the leymus chinensis and provides a direct molecular target for directionally creating new varieties of pasture and cereal crops with high fruiting rate.

Inventors

MA XIQING
LIU ZHENZHEN
MA SHU
WEI JIA
BI LEI
ZHU RUONAN
Lei Mengzhou
XU XINXIN

Assignees

中国农业大学

Dates

Publication Date: 20260512
Application Date: 20260228

Claims (10)

Application of LcCBP60b gene in regulating growth and development of leymus chinensis and setting rate; Wherein, lcCBP b gene is: i) A nucleotide sequence shown in SEQ ID NO. 1; ii) the nucleotide sequence shown in SEQ ID NO. 1 is substituted, deleted and/or added with one or more nucleotides and expresses the same functional protein; iii) The nucleotide sequence which hybridizes with the sequence shown in SEQ ID No. 1 and expresses the same functional protein under stringent conditions, i.e., in a 0.1 XSSPE solution containing 0.1% SDS or 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution, or, Iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.
2. The use according to claim 1, wherein the modulation is negative.
3. The method according to claim 2, wherein the method comprises inhibiting the expression of LcCBP b gene or the activity of the coded protein thereof to increase the fruiting rate of leymus chinensis and/or increase the spike length and spike number.
4. A method for increasing the maturing rate, the spike length and the spike number of leymus chinensis, which is characterized in that the method comprises the steps of reducing the expression level of LcCBP b gene or the activity of coded protein in leymus chinensis; Wherein the LcCBP b gene is as described in claim 1.
5. The method according to claim 4, wherein the substance for reducing the expression level of LcCBP b gene or the activity of the protein encoded thereby in leymus chinensis is at least one selected from shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA/DNA, low molecular weight compounds, peptides, and antibodies.
6. The method of claim 4, wherein the expression level of LcCBP b gene is reduced using an RNA interference method.
7. The method of claim 6, wherein LcCBP b gene silencing expression vector is introduced into the leymus chinensis such that the expression level of LcCBP b gene in the transgenic leymus chinensis plant is reduced compared to non-transgenic wild type leymus chinensis; Wherein the LcCBP b gene silencing expression vector comprises a nucleic acid molecule capable of specifically inhibiting the expression of the leymus chinensis LcCBP b gene, and the nucleic acid molecule comprises a sequence complementary to a nucleotide sequence shown in SEQ ID NO. 1 or a fragment thereof.
8. The method of claim 7, wherein the nucleic acid molecule consists of 1-250bp of the nucleotide sequence set forth in SEQ ID NO. 1.
The application of the LcCBP60b-LcALY2 molecular module in regulating and controlling the growth and the setting rate of the leymus chinensis is characterized in that the growth and the setting rate of the leymus chinensis are regulated and controlled through regulating and controlling the interaction of LcCBP b protein and LcALY2 protein; wherein the LcCBP b protein is encoded by the LcCBP b gene of claim 1; The LcALY protein is encoded by LcALY2 gene, and the LcALY gene is: i) A nucleotide sequence shown in SEQ ID NO. 2; II) the nucleotide sequence shown in SEQ ID No. 2 is substituted, deleted and/or added with one or more nucleotides and expresses the nucleotide sequence of the same functional protein; III) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID No. 2 and expresses the same functional protein under stringent conditions, either in a 0.1 XSSPE solution containing 0.1% SDS or in a 0.1 XSSC solution containing 0.1% SDS, at 65℃and washing the membrane with the solution, or, IV) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of I), II) or III) and expresses the same functional protein.
10. The use according to claim 9, wherein the expression or activity of LcCBP b protein is reduced to weaken the binding of LcCBP b protein and LcALY b protein, so as to release the transcription inhibition of downstream flowering and embryo development related genes, and further improve the fruiting rate of leymus chinensis and/or increase the spike length and spike number.

Description

Application of LcCBP b-LcALY2 molecular module in regulation and control of growth and development of leymus chinensis and setting rate Technical Field The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of LcCBP b-LcALY2 molecular modules in regulation and control of growth and development of leymus chinensis and fruiting rate. Background The leymus chinensis is perennial rhizome type perennial pasture of Gramineae, is a dominant grass seed of European Asia grassland, is widely distributed in northeast and inner Mongolia grasslands, north China and northwest areas of China, and has the advantages of high yield, high protein content, good palatability, strong regeneration capacity and wide adaptability. As a main colonisation seed of temperate grasslands, the leymus chinensis can realize rapid growth and reproduction by virtue of developed root systems, and can survive for 20 years at most, thus being an excellent pasture variety for sand control and ecological protection. The leymus chinensis has the advantages of long service life, strong asexual reproduction capability and the like, but has poor sexual reproduction capability, and is mainly characterized by low heading rate, low fruiting rate and low germination rate, thus severely restricting the application and popularization of the leymus chinensis. The molecular mechanism of the burial character of the leymus chinensis is excavated, and the cultivation of leymus chinensis varieties with high burial rate is a primary task for realizing the jump of production and application of the leymus chinensis. At present, researchers mainly develop researches around main factors influencing the fruiting character of the leymus chinensis. Studies show that the fruiting character of the leymus chinensis is influenced by the germplasm type and the cultivation condition of the leymus chinensis, and inflorescence parts, opening time, spike development conditions and pollen formation conditions of different leymus chinensis germplasm are different. Meanwhile, different sheep germplasm resources are influenced by different ecological environment conditions, the heading rate and the fruiting rate are relatively different, and the variation range is respectively up to 2.1-71.3% and 7.8-73.3%. The study and investigation of the flowering sequence, the flowering period length, the florescence time and the like of 5 sheep grass germplasm find that a plurality of characters in different sheep grass germplasm have larger difference, wherein the florescence time can last to open for 2 hours at the longest. Meanwhile, gibberellin and kinetin are sprayed in the green-turning stage and the tillering stage of the leymus chinensis, the spike number, the floret number, the fruiting rate and the spike weight of the leymus chinensis can be remarkably improved, the photosynthesis of the leymus chinensis can be improved through exogenous application of CO 2, the climatic stage of the leymus chinensis is affected, and the heading of the leymus chinensis is promoted. However, the present situation of the fruiting of the leymus chinensis is explored based on a single factor, the stability of the high fruiting rate character of the leymus chinensis is difficult to maintain, and the leymus chinensis is limited by equipment and cost. Therefore, the development of molecular mechanism research of the fruiting character of the leymus chinensis is expected to be capable of cultivating a new leymus chinensis variety with high fruiting quality and stability through molecular breeding. The calmodulin binding protein (Camodulin-binding protein 60) family is a family of atypical transcription factors characteristic of plants. Earlier studies found that the distribution of 8 family members, CBP60a-g and SARD1, in the arabidopsis genome, wherein CBP60g and SARD1 play a key role in the immune response of plants, which enhances the plant's resistance to pathogenic bacteria mainly by upregulating the salicylic acid pathway of the plant. More and more research results show that calmodulin binding protein and its pathway related gene play important roles in the growth and development of plants and the like. Under the condition of short sunlight, gibberellin activity and calcium ion content in chrysanthemum are increased, and a complex is formed by calcium signal transduction key factor calmodulin CmCAM and gibberellin signal key inhibitor CmGAI, so that inhibition on CmGAST1 is relieved, and chrysanthemum flowering is promoted. However, the specific mechanism of the calmodulin binding protein and the related pathways for regulating and controlling the fruiting character of the leymus chinensis is not clear at present. Disclosure of Invention The invention aims to provide an application of LcCBP b-LcALY2 molecular module in regulating and controlling growth and development of leymus chinensis and setting rate. In order to achieve the aim of the inv