Cloning and application of multi-effect gene ccpprr7 controlling flowering time, plant height and fiber yield of corchorus capsuarius

Abstract

This invention belongs to the field of molecular breeding technology and relates to a multi-effect gene that controls the flowering period, plant height, and fiber yield of jute. CcPRR7 The cloning and application of *Arabidopsis thaliana*. The steps were as follows: A QTL controlling flowering time, plant height, and fiber yield was located on chromosome 3 through genome-wide association analysis. Candidate regions were narrowed down using linkage disequilibrium, and all genes within these regions were extracted and functionally annotated, leading to the discovery of a *Arabidopsis thaliana* QTL. PRR7 The direct homologous gene was named CcPRR7 Build CcPRR7 Overexpression vectors were used to transform Arabidopsis thaliana, and the flowering period of transgenic positive plants was 8 days later than that of wild-type plants. This invention provides gene resources for the genetic improvement and molecular breeding of jute.

Description

Technical Field

[0001] This invention belongs to the field of molecular breeding technology and relates to the cloning and application of the pleiotropic gene CcPRR7, which controls the flowering period, plant height and fiber yield of jute. Background Technology

[0002] Jute is an annual secondary bast fiber crop belonging to the genus *Corchorus* L. of the Malvaceae family (Malvaceae). Its fiber production accounts for 80% of the world's total bast fiber production. The genus *Corchorus* contains over 100 species, with *Corchorus capsularis* and *Corchorus olitorius* being of cultivation value. These two species have different growth habits and reproductive isolation. Jute thrives in warm, humid environments. Its main producing countries are China, Bangladesh, and India, with smaller-scale cultivation in other countries such as Egypt, Nepal, Zimbabwe, and Thailand. Globally, jute's production and planting area are second only to cotton among fiber crops, making it an important raw material for the bast fiber industry.

[0003] Flowering period, plant height, and fresh husk weight per plant are all important agronomic traits related to jute fiber yield. As a typical short-day crop, the main controlling factor for jute's flowering period is photoperiod; it requires less than 12 hours of light per day to flower. If the light duration exceeds this, flowering will be delayed or even absent. Jute is a light-loving crop and is very sensitive to light, with long-fruited jute varieties being more sensitive than round-fruited varieties. Premature flowering in jute leads to a lower first branching point, more branching, thinner husks, shorter plants, and a significant decrease in yield and quality. Plant height is one of the most important traits directly determining jute fiber yield. It exhibits rich variation in wild germplasm resources and is an essential trait that needs to be considered when breeding superior varieties. Fresh husk weight per plant is an important indicator of jute yield, mainly depending on the accumulation of biomass during the vegetative growth period. A longer vegetative growth period results in a higher fresh husk weight per plant, directly reflecting jute fiber yield.

[0004] Therefore, discovering and cloning the pleiotropic gene CcPRR7 is urgent and necessary for developing pleiotropic genes for jute flowering period, plant height, and fiber yield with Chinese intellectual property rights. Summary of the Invention

[0005] The purpose of this invention is to provide a multi-effect gene and its sequence that controls the flowering period, plant height and fiber yield of jute, laying the foundation for the genetic improvement of jute.

[0006] The technical solution implemented by this invention: The specific steps of this invention are as follows: (1) The flowering period, plant height, and fresh husk weight of 299 jute germplasm resources were investigated and recorded.

[0007] (2) Resequencing of 299 jute germplasm resources.

[0008] (3) Filter and screen SNP sites, and perform genome-wide association analysis on the three traits respectively.

[0009] (4) The QTL interval of chromosome 3 was determined to be between 5.45Mb and 6.25Mb.

[0010] (5) The candidate interval was narrowed down to between 5.45Mb and 5.78Mb by using the chain imbalance.

[0011] (6) Extract the protein sequences of all genes in this region according to the jute genome annotation file and perform Blastp alignment.

[0012] (7) Among them, Cc.03G0003190 is homologous to Arabidopsis thaliana PRR7 and is identified as the candidate gene CcPRR7. The CDS sequence of the CcPRR7 gene is shown in Seq ID NO.2, and its encoded amino acid sequence is shown in Seq ID NO.1.

[0013] A plant expression vector containing the aforementioned CcPRR7 gene.

[0014] A host bacterium containing the aforementioned CcPRR7 gene.

[0015] The aforementioned CcPRR7 gene, or plant expression vector, or host bacteria are used to control the flowering period, plant height, and fiber yield of jute.

[0016] The aforementioned CcPRR7 gene, or plant expression vector, or host bacteria are used in the genetic improvement and molecular breeding of jute.

[0017] The aforementioned CcPRR7 gene, or plant expression vector, or host bacteria are used to delay the flowering period of Arabidopsis thaliana. Attached Figure Description

[0018] Figure 1 Manhattan plot and quantile plot of genome-wide association analysis of jute flowering period.

[0019] Figure 2 Manhattan plot and quantile plot of genome-wide association analysis of jute plant height.

[0020] Figure 3 Manhattan plot and quantile plot of genome-wide association analysis of fresh husk weight of jute single plant.

[0021] Figure 4 Transgenic Arabidopsis thaliana phenotype. Detailed Implementation

[0022] The present invention will be further illustrated below through embodiments, the purpose of which is only to better understand the content of the present invention and not to limit the scope of protection of the present invention.

[0023] All raw materials and equipment used in this invention are commercially available products that can be directly purchased from the market, and the primer sequences used are synthesized by Fuzhou Platinum Technology Co., Ltd.

[0024] Example 1: Genome-wide association analysis of flowering time, plant height, and single-plant fresh husk weight in jute 1) Record the flowering period, plant height, and fresh husk weight per plant of 299 jute germplasm resources. 299 jute germplasm resources were sown in the experimental field, planted in two-row plots with a row length of 3.5m and a row spacing of 1.2m × 0.1m. A single-factor randomized block design was used, with two protective rows planted along the perimeter. Field management was the same as in the main field. After the first flower appeared in each plot, observations were taken every other day from 9:00 to 10:00 AM. All plants in the experimental plot were observed, and the date when 50% of the plants were in bloom was recorded as the flowering period. When the plants in the plot reached the technological maturity stage, 10 plants were randomly selected to measure their height, and the average value was recorded. The bark of 10 jute plants was then removed, and the fresh bark weight was measured using an electronic balance. The average value of these fresh bark weights was recorded as the single-plant fresh bark weight. The fiber yield can be measured using the single-plant fresh bark weight.

[0025] 2) Resequencing of 299 jute germplasm resources 299 jute germplasm resources were resequencing and aligned to a reference genome. Low sequencing depth, repetitive or heterozygous sites were filtered out, and a total of 3,415,772 SNP markers were used for subsequent analysis.

[0026] 3) Genome-wide association analysis Genome-wide association analysis (GLM) was performed using the General Linear Model (GLM) in Tassel 5 software, locating a QTL controlling flowering time, plant height, and fiber yield in jute on chromosome 3. Candidate regions were narrowed down using linkage disequilibrium, and all genes within these regions were extracted and functionally annotated, revealing an orthologous gene of Arabidopsis thaliana PRR7, which was named CcPRR7. Manhattan plots and quantile plots were visualized using the CMplot package in RStudio software; see [link to documentation]. Figure 1 , Figure 2 and Figure 3 .

[0027] Example 2: Cloning and sequence analysis of the CDS of the Jute CcPRR7 gene 1) RNA extraction Young leaves of the jute variety "Jute 179" were taken, frozen and ground into powder in a ceramic mortar with liquid nitrogen, and then RNA was extracted using the OMEGA kit procedure and stored at -80℃ for later use.

[0028] 2) Cloning of the CcPRR7 gene CDS RNA frozen at -80℃ was retrieved, and cDNA first strand was synthesized according to the TaKaRa reverse transcription kit procedure. The cDNA was then stored at -20℃. Two primers were designed as follows: CcPRR7-CDS-F (Seq ID NO.3) and CcPRR7-CDS-R (Seq ID NO.4). The PCR reaction system consisted of: 10 μL cDNA template, 2.5 μL each of forward and reverse primers (10 μM), 20 μL of mix (Tiangen Biotech Beijing Co., Ltd., KT201), and ddH2O added to a final volume of 50 μL. Amplification conditions were: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, 34 cycles; 72℃ final extension for 7 min; and storage at 4℃. The PCR product was sent to a company for sequencing analysis. The CDS sequence of the CcPRR7 gene is shown in Seq ID NO.2, and the amino acid sequence of the encoded protein is shown in Seq ID NO.1.

[0029] Example 3: Transformation of Arabidopsis thaliana with jute CcPRR7 overexpression vector 1) Construction of CcPRR7 overexpression vector Double digestion was performed on the SacI (5' end) and BamHI (3' end) restriction sites selected from the multiple cloning site of pCAMBIA1300S, followed by gel extraction to obtain the linear vector. Based on the vector information and restriction sites, adapter primers CcPRR7-adaptor-F (Seq ID NO. 5) and CcPRR7-adaptor-R (Seq ID NO. 6) were designed at both ends of the full-length CDS of the CcPRR7 gene. PCR amplification was performed to obtain the target fragment, which was then recovered from the gel. The linear vector and target fragment were ligated using in-fusion to obtain the recombinant plasmid. The recombinant plasmid was transformed into DH5α E. coli competent cells using the heat shock method and plated on LB agar plates containing kanamycin resistance. The cells were incubated overnight at 37°C for 12–16 h. The following day, single clones were selected and subjected to bacterial culture PCR using specific primers CcPRR7-OV detection-F (Seq ID NO.7) and CcPRR7-OV detection-R (Seq ID NO.8). Samples with positive bands were sent to the company for sequencing. After the sequencing was confirmed to be correct, plasmids were extracted and stored at -20°C.

[0030] 2) Arabidopsis genetic transformation The constructed CcPRR7 overexpression vector was transformed into Arabidopsis plants using Agrobacterium-mediated inflorescence transfection. After 24 hours of dark culture, the plants were transferred to light conditions for normal growth. Mature seeds of the T1 generation were harvested approximately three weeks later. The T1 generation seeds were then cultured on a medium containing hygromycin for selection, and positive T1 generation seedlings were selected. These seedlings were then cultured further to harvest T2 generation seeds. The T2 generation seeds were then sown on a medium containing hygromycin, and DNA from viable lines was extracted using the CTAB method. Positive plants were then selected by PCR using primers CcPRR7-OV detection-F (Seq ID NO. 7) and CcPRR7-OV detection-R (Seq ID NO. 8). A positive band indicated a transgenic line.

[0031] The flowering time of 10 wild-type Arabidopsis thaliana plants and 20 transgenic Arabidopsis thaliana plants was investigated. The results showed that the average time from sowing to the opening of the first flower after bolting in wild-type Arabidopsis thaliana was 28.3 days; the average flowering time of the overexpressing transgenic lines was 36.4 days, approximately 8 days later. Figure 4 The result was statistically significant (p-value less than 0.05).

Claims

1. A pleiotropic gene controlling flowering time, plant height, and fiber yield in jute. CcPRR7 Its features are: The pleiotropic gene CcPRR7 The CDS sequence is shown in Seq ID NO.2, and the amino acid sequence it encodes is shown in Seq ID NO.

1.

2. A method for cloning the pleiotropic gene as described in claim 1. CcPRR7 The primer pair is characterized by: The nucleotide sequences of the primer pairs are shown in Seq ID NO.3 and Seq ID NO.

4.

3. A gene comprising the pleiotropic gene as described in claim 1 CcPRR7 Plant expression vectors.

4. A gene comprising the pleiotropic gene as described in claim 1 CcPRR7 The host bacteria.

5. The pleiotropic gene as described in claim 1 CcPRR7 The application of the plant expression vector as described in claim 3, or the host bacteria as described in claim 4, in delaying the flowering period of jute through overexpression.

6. The pleiotropic gene as described in claim 1 CcPRR7 The application of the plant expression vector as described in claim 3, or the host bacteria as described in claim 4, in jute genetic improvement and molecular breeding aimed at delaying the flowering period of jute through overexpression.

7. The pleiotropic gene as described in claim 1 CcPRR7 The application of the plant expression vector as described in claim 3, or the host bacteria as described in claim 4, in delaying the flowering period of Arabidopsis thaliana through overexpression.

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