A castor bean PIP5K11 gene and its application

Abstract

This invention discloses a castor bean PIP5K11 gene and its applications, relating to the field of plant genetic engineering technology. The key technical point is that the full-length nucleotide sequence of the PIP5K11 gene is SEQ ID No. 1, encoding 395 amino acids. Overexpression of the PIP5K11 gene can promote earlier flowering, fruit ripening, and improved seed quality, while inhibiting the growth of primary branches and the development of flowers and fruits. The PIP5K11 gene also affects the expression levels of its family members, PIP5K1, PIP5K2, PIP5K6, PIP5K8, and PIP5K9 genes.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to a castor bean PIP5K11 gene and its applications. Background Technology

[0002] Castor bean (Ricinus communis L.) is an annual or perennial dicotyledonous herbaceous plant belonging to the genus Ricinus in the family Euphorbiaceae. Studies have revealed its numerous applications, leading to its use in various fields. The inflorescence development of castor beans mainly falls into two categories: one, the vast majority of castor beans worldwide are female lines, exhibiting both monoecious (female) and hermaphroditic (ectophobic) inflorescences; and two, developed in 1990 by the Tongliao Academy of Agricultural Sciences in Inner Mongolia through 60Coγ mutagenesis, resulting in the Lm-type female castor bean line (also known as the marker female line). The growth and development of the castor bean inflorescence is a crucial factor affecting yield, and studying its development is of great significance for improving castor bean yield per unit area.

[0003] Phosphatidylinositol 4-phosphate 5-kinase (PIP5K) is a phospholipid kinase in organisms that catalyzes the phosphorylation of PI-4-phosphate (PI4P) to synthesize PI-4,5-bisphosphate [PI(45)P2], and is a key enzyme in the phosphatidylinositol (PI) signal transduction pathway. Phosphatidylinositol 4,5-bisphosphate can regulate actin cytoskeleton, vesicle transport, etc. The PIP5K gene family regulates plant growth and development in the phosphatidylinositol signal transduction pathway, including gene expression in plant cells, cellular responses to external environmental factors, regulation of ion channels, and hormone action. Currently, there is relatively little research on PIP5K family genes in castor beans. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention uses castor bean Lm-type female lines as material and provides the biological application of the PIP5K11 gene in the inflorescence development process of castor bean Lm-type female lines through homologous overexpression and gene knockout.

[0005] The present invention provides the following technical solution: a castor bean PIP5K11 gene, wherein the full-length nucleotide sequence of the PIP5K11 gene is SEQ ID No.1, and the gene encodes 395 amino acids.

[0006] Furthermore, the PIP5K11 gene is used in plant functional gene research or in plant genetic engineering breeding.

[0007] Furthermore, the overexpression of the PIP5K11 gene can be applied in any aspect of promoting early flowering, fruit ripening, improving seed quality, inhibiting the growth of primary branches, and the development of flowers and fruits.

[0008] Furthermore, the overexpression of the PIP5K11 gene is used to promote pollen viability or increase pollen germination rate.

[0009] Furthermore, when the PIP5K11 gene was overexpressed, the expression levels of PIP5K1 and PIP5K9 were significantly downregulated, while the expression levels of PIP5K2, PIP5K6, and PIP5K8 were significantly upregulated; when the PIP5K11 gene was knocked out, the expression levels of PIP5K1, PIP5K2, PIP5K6, PIP5K8, and PIP5K9 were all significantly downregulated.

[0010] Furthermore, the application of the PIP5K11 gene in plant functional gene research or in plant genetic engineering breeding includes the following steps:

[0011] (1) Construct overexpression or knockout vectors, and use homologous recombination to transform the resulting recombinant plasmids into Agrobacterium competent cells.

[0012] (2) By using Agrobacterium-mediated transformation, the constructed vector containing the target gene is transferred into the cotyledonary node of the plant. Plants with overexpression or knockout of the target gene are screened, and then domesticated and transplanted.

[0013] Furthermore, the procedure also includes the following steps: extracting total RNA from overexpressing or knockout resistant plants, reverse transcribing it into cDNA, performing real-time quantitative PCR using the cDNA as a template, and measuring the expression level of the target gene by RT-qPCR.

[0014] Further, in step (1), the overexpression vector is pBI121-3*flag, and the cloning primer sequence is: GPIP5K11-F:

[0015] 5′-ACGGGGGACTCTAGAGGATCCATGGCGGAGTCAAGCGAGA-3′;

[0016] GPIP5K11-R:

[0017] 5′-GTCCCGGTACCCCCGGGGATCCCAGATTTTGGCAGGGTAGATCAG-3′.

[0018] Further, in step (1), the knockout vector is PKSE401, and the primer sequences for the dual-target sgRNA are:

[0019] PIP5K11-gRNA1: 5′-CTTCATTTCAGATTTTCGCAGGG-3′;

[0020] PIP5K11-gRNA2: 5′-GCAAAGTTACCGAGTTAGAATGG-3′.

[0021] In summary, the present invention has the following beneficial effects:

[0022] This invention provides a castor bean PIP5K11 gene, the full-length nucleotide sequence of which is SEQ ID No. 1. This gene encodes 395 amino acids. Through homologous recombination, overexpression and knockout vectors were constructed, and it was found that the PIP5K11 gene can be used for plant functional gene research or in plant genetic engineering breeding. Overexpression of the PIP5K11 gene in resistant plants promotes earlier flowering and fruit ripening in castor beans, improves seed quality, and inhibits the growth of primary branches and the development of flowers and fruits. Furthermore, the seeds of the PIP5K11 gene overexpression resistant plants G11-1 and G11-2 germinate faster, have a higher germination rate, and have longer radicles. PIP5K11 gene knockout-resistant plants delay castor bean flowering, seed maturation, and reduce seed quality. It also inhibits the growth of primary branches and the development of flowers and fruits; pollen viability is weak, mature pollen germination is weak, and ovary development and fruit formation are affected. Furthermore, the PIP5K11 gene affects the expression levels of its family members, PIP5Ks: when the PIP5K11 gene is overexpressed, the expression levels of PIP5K1 and PIP5K9 are significantly downregulated, while the expression levels of PIP5K2, PIP5K6, and PIP5K8 are significantly upregulated; when the PIP5K11 gene is knocked out, the expression levels of PIP5K1, PIP5K2, PIP5K6, PIP5K8, and PIP5K9 are all significantly downregulated. Attached Figure Description

[0023] Figure 1 This is an electrophoresis image of PCR amplification of the castor bean PIP5K11 gene. M: DL 5000 Marker; 1 and 2: PIP5K11.

[0024] Figure 2 This is an electrophoresis image of the PCR amplification of the E. coli recombinant vector pBI121-3*flag-PIP5K11 from Example 2;

[0025] Figure 3 These are PIP5K11 gene overexpression resistant plants obtained in Example 2;

[0026] Figure 4 This is the RT-qPCR detection result of the PIP5K11 gene overexpressed in the resistant plant of Example 2;

[0027] Figure 5 This is a comparison diagram of seed germination between PIP5K11 gene overexpression resistant plants and wild-type seeds in Example 2;

[0028] Figure 6 This is an agarose gel electrophoresis image of the PCR of the Agrobacterium knockout recombinant plasmid from Example 3;

[0029] Figure 7 This is the Cas9-PIP5K11 sequencing alignment diagram from Example 3, where A: sequence alignment of the knockout resistant plant Q11-1; B: sequence alignment of the knockout resistant plant Q11-2.

[0030] Figure 8 This is a bar chart of RT-qPCR detection in PIP5K11 gene knockout resistant plants from Example 3;

[0031] Figure 9 This is an observation diagram of pollen germination rate in PIP5K11 gene knockout plants from Example 3;

[0032] Figure 10 This is a bar chart showing the expression levels of other family members PIP5K1, PIP5K2, PIP5K6, PIP5K8, and PIP5K9 genes after overexpression of the PIP5K11 gene in Example 4.

[0033] Figure 11 This is a bar chart showing the expression levels of the PIP5K1, PIP5K2, PIP5K6, PIP5K8, and PIP5K9 genes, which are other members of the PIP5K11 gene family after knockout in Example 4. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the embodiments.

[0035] The castor bean material used in the following examples is the aLmAB2 strain of the Lm type female line, provided by the Key Laboratory of Castor Bean Breeding of the State Ethnic Affairs Commission; the Escherichia coli DH5α strain and Agrobacterium tumefaciens GV3101 strain were ordered by Beijing Zhuangmeng International Biotechnology Co., Ltd.; the CRISPR / Cas9 knockout vector pKSE401 was ordered by Shanghai Kaiyi Biotechnology Co., Ltd.

[0036] Example 1: Cloning of the PIP5K11 gene from castor bean

[0037] Seamless cloning primers were designed using CE Design V1.03 software. The CDS sequence of the PIP5K11 gene was selected, and the pBI121-3*flag vector was constructed using plant overexpression. The primer sequence was: GPIP5K11-F.

[0038] 5′-ACGGGGGACTCTAGAGGATCCATGGCGGAGTCAAGCGAGA-3′;

[0039] GPIP5K11-R:

[0040] 5′-GTCCCGGTACCCCCGGGGATCCCAGATTTTGGCAGGGTAGATCAG-3′;

[0041] Reaction system: 5×primer star buffer (Mg 2+ The pBI121-GFP-PIP5K11 *E. coli* strain was prepared by plasmid extraction using 10.0 μL of dNTP Mixture (2.5 μM), 1.0 μL of Primer F, 1.0 μL of Primer R, 3.0 μL of DNA Templet, 0.5 μL of 2.5 U / μL Primerstar HS DNA polymerase, and 0.5 μL of ddH2O3.

[0042] Example 2: Overexpression of the PIP5K11 gene in castor bean

[0043] 1. PIP5K11 gene cloning

[0044] The same as the castor bean PIP5K11 gene clone in Example 1.

[0045] 2. Construction of PIP5K11 gene overexpression vector

[0046] The overexpression vector pBI121 was ligated with a 3*flag tag: the pBI121 plasmid vector was digested with restriction endonuclease KpnIⅠ. The 3*flag gene fragment was amplified using fusion PCR technology, with the primer sequences as follows:

[0047] 3*flag-F: 5′-CGGACTACAAAGACCATGACGGTGATTATAAAGATCAT GACATCGATTACAAGGATGACGATGACAAGTGAGGTAC-3′;

[0048] 3*flag-R: 5′-CTCACTTGTCATCGTCATCCTTGTAATCGATGTCATG ATCTTTATAATCACCGTCATGGTCTTTGTAGTCCGGTAC-3′;

[0049] The primers were self-crossed at a 1:1 ratio, and PCR was performed using the following program: denaturation at 94℃ for 10 min; annealing at 65℃ for 10 min; extension at 37℃ for 10 min; further extension at 25℃ for 10 min; and storage at 4℃. The 3*flag gene fragment was obtained and ligated into the purified, linearized pBI121 vector using T4 DNA ligase at a molar ratio of 3:1. The resulting recombinant plasmid was named pBI121-3*flag. The vector containing 3*flag was transformed into competent DH5α cells of *E. coli*. The target gene fragment PIP5K11 was ligated into the pBI121-3*flag vector using a seamless cloning method. The ligation product containing the target gene was transformed into *E. coli*, and the resulting recombinant plasmid was named pBI121-3*flag-PIP5K11. Plasmids were extracted from correctly sequenced *E. coli* bacterial cultures using a plasmid mini-extraction kit to obtain the recombinant plasmid pBI121-3*flag-PIP5K11. The recombinant plasmid pBI121-3*flag-PIP5K11 was transformed into *Agrobacterium* competent GV3101 cells. Using the Lm-type female castor bean line aLmAB2 as material, the constructed overexpression vector containing the target gene was transformed into the cotyledonary nodes of castor bean using *Agrobacterium*-mediated transformation. Plants resistant to target gene overexpression were screened, domesticated, and transplanted to obtain PIP5K11 gene overexpression resistant plants G11-1 and G11-2. Wild-type plant WT-1 was used as a control, and molecular and biological identifications were performed on them, respectively.

[0050] At 90 days after transplanting, the wild-type plant WT-1 experienced less basal leaf drop, while the PIP5K11 overexpression resistant plants G11-1 and G11-2 showed faster basal leaf senescence. WT-1 had not yet developed inflorescences, while G11-1 and G11-2 showed inflorescences and flowered earlier. At 120 days after transplanting, the wild-type plant WT-1 showed primary branching, and the main stem spike was just flowering, while the PIP5K11 overexpression resistant plants G11-1 and G11-2 had not yet developed primary branching, but their inflorescences had already completed pollination and entered the fruit development stage. Compared with WT-1, G11-1 flowered about 30 days earlier, and G11-2 flowered about 20 days earlier. The capsule of G11-1 had 4 locules, while the capsule of G11-2 had 3 locules, and the splitting of G11-1 and G11-2 was more pronounced than that of WT-1. The seeds of G11-1 and G11-2, after maturation, were fuller, darker in color, and had more distinct and glossy seed coat patterns compared to WT-1. These results indicate that, compared to the wild-type control, PIP5K11 gene overexpression resistant plants promote earlier flowering and fruit maturation in castor beans, improve seed quality, and simultaneously inhibit the growth of primary branches and the development of flowers and fruits.

[0051] When the same number of mature seeds from PIP5K11 gene-overexpressing resistant plants G11-1 and G11-2 and wild-type plant WT-1 were germinated for 72 hours, the seeds of PIP5K11 gene-overexpressing resistant plants G11-1 and G11-2 germinated faster and had a higher germination rate than wild-type plant WT-1. Furthermore, the radicles of PIP5K11 gene-overexpressing resistant plants G11-1 and G11-2 were longer.

[0052] Example 3: CRISPR / Cas9 knockout of the PIP5K11 gene in castor bean

[0053] Based on the provided DNA and RNA sequences, sgRNA sequences were selected using a gRNA design website to design dual-target sgRNAs. The primer sequences are as follows:

[0054] PIP5K11-gRNA1: 5′-CTTCATTTCAGATTTTCGCAGGG-3′;

[0055] PIP5K11-gRNA2: 5′-GCAAAGTTACCGAGTTAGAATGG-3′;

[0056] The PKSE401 vector and the complete gene were digested with BsaI to synthesize double sgRNA target primers. Ligation was performed overnight at 4°C using T4 DNA ligase. The ligation product was transformed into competent *E. coli* DH5α cells via heat shock, plated on LB agar plates containing 50 mg / L kanamycin, and incubated overnight at 37°C on a shaker at 180 rpm for transformation resistance screening. Four to five single colonies were picked and cultured in 100 μL of liquid LB medium for 2 hours. PCR detection was then performed using primers designed with the PKSE401 vector. The PCR products were analyzed by 1% agarose gel electrophoresis. Approximately 800 μL of the bacterial culture with the correct band size was mixed thoroughly with approximately 200 μL of autoclaved glycerol to obtain the complete PIP5K11 gene knockout vector PKSE401-PIP5K11-gRNA. The knockout expression vector plasmid was transformed into Agrobacterium GV3101. Using the Lm-type female castor bean strain aLmAB2 as material, the constructed knockout vector containing the target gene was transferred into the cotyledonary nodes of castor beans via Agrobacterium-mediated transformation. Knockout resistant plants were screened, domesticated, and transplanted, resulting in knockout resistant plants Q11-1 and Q11-2. Total RNA was extracted from the inflorescence axes of the PIP5K11 knockout resistant plants Q11-1 and Q11-2, as well as the wild-type plant WT-2, during the flowering stage. This RNA was reverse transcribed into cDNA, and real-time quantitative PCR was performed using the cDNA as a template. RT-qPCR identification was then performed. Figure 8It can be seen that, in the inflorescence axis, the expression level of PIP5K11 gene knockout resistant plants Q11-1 and Q11-2 was significantly downregulated compared with wild-type plant WT-2.

[0057] By comparing the functional domain target sequences, it was found that in the PIP5K11 gene knockout resistant plant Q11-1, the GTT base on the gRNA1 chain at the target site was replaced with GCT, and valine (Val) was changed to alanine (Ala). A frameshift mutation was also observed in the functional domain A of the gRNA1 chain at the target site. Similarly, in another PIP5K11 gene knockout resistant plant, Q11-2, a frameshift mutation was also observed in the functional domain A of the gRNA1 chain at the target site. Both PIP5K11 gene knockout resistant plants Q11-1 and Q11-2 exhibited hermaphroditic inflorescences.

[0058] At 90 days after transplanting, compared with the wild-type plant WT-2, the PIP5K11 gene knockout resistant plants Q11-1 and Q11-2 were taller and had fewer basal leaves falling off; WT-2 showed inflorescences, while Q11-1 and Q11-2 had not yet shown inflorescences, and flowering was delayed. At 120 days after transplanting, WT-2 showed primary branching, and the inflorescences had completed pollination and entered the fruit development stage, while G11-1 and G11-2 had not yet shown primary branching, and the main stem spikes were only flowering. Compared with WT-2, Q11-1 flowered about 25 days later, and Q11-2 flowered about 35 days later. After pollination, most of the female flowers of Q11-1 and Q11-2 failed to develop into fruits and withered. During the later stages of castor bean inflorescence development, the capsules of Q11-1, Q11-2, and WT-2 all had three locules. Compared to WT-2, the capsules of Q11-1 and Q11-2 showed less obvious splitting and were smaller in size. After maturation, the seeds of Q11-1 and Q11-2 were smaller, wrinkled, and had a shallower seed coat pattern and lacked luster compared to WT-2. These results indicate that, compared to the wild-type control, the PIP5K11 gene knockout resistant plants delayed flowering, seed maturation, and reduced seed quality in castor beans, while also inhibiting the growth of primary branches and the development of flowers and fruits.

[0059] By measuring the viability of mature castor pollen, stronger viability resulted in deeper staining, while weaker viability resulted in colorless staining. Observing 100 pollen grains in different fields of view, the PIP5K11 gene knockout resistant plants Q11-1 and Q11-2 showed weaker pollen viability compared to the wild-type plant WT-2. The percentage of viable pollen in Q11-1 was 21%, while the percentage in Q11-2 was 33%. This indicates that the knockout of the PIP5K11 gene affects the pollination and fertilization process of castor beans, thus impacting plant yield.

[0060] The germination rate of mature pollen in PIP5K11 gene knockout resistant plants was measured. One hundred pollen grains were observed and counted in different fields. Compared with the wild-type plant WT-2, the mature pollen germination rate of PIP5K11 gene knockout resistant plants Q11-1 and Q11-2 was weaker in Q11-1 and Q11-2. The percentage of pollen tubes germinating from Q11-1 pollen was 23%, while the percentage from Q11-2 pollen tubes was 30%. This indicates that PIP5K11 gene knockout affects the germination of viable pollen in castor beans, leading to pollen tube formation and consequently affecting ovary development and fruit formation.

[0061] Example 4: Effect of PIP5K11 gene on the expression level of PIP5Ks in the same family

[0062] The expression levels of other members of the PIP5K gene family, namely PIP5K1, PIP5K2, PIP5K6, PIP5K8, and PIP5K9, in the PIP5K11 gene overexpression resistant plants G11-1 and G11-2 and the PIP5K11 gene knockout resistant plants Q11-1 and Q11-2 were analyzed using RT-qPCR. The 18S gene was selected as the internal reference gene, and the specific primer sequences for the above genes are shown in Table 1 below.

[0063] Table 1. RT-qPCR primer sequences for other PIP5Ks member genes.

[0064]

[0065] Five biological replicates were performed for each experimental material, and the mean CT value was used. The relative expression levels of other PIP5Ks member genes were calculated using the 2-ΔΔCt method. SPSS 19.0 software was used for data statistics and analysis. Significance analysis was performed using the independent samples t-test for comparing means. The results are presented as a bar chart of relative expression levels as shown below. Figure 10 and Figure 11 As shown.

[0066] Depend on Figure 10-11 It can be seen that the PIP5K11 gene affects the expression levels of PIP5Ks in the same family. When the PIP5K11 gene is overexpressed, the expression levels of PIP5K1 and PIP5K9 are significantly downregulated, while the expression levels of PIP5K2, PIP5K6, and PIP5K8 are significantly upregulated. When the PIP5K11 gene is knocked out, the expression levels of PIP5K1, PIP5K2, PIP5K6, PIP5K8, and PIP5K9 are all significantly downregulated.

[0067] This specific embodiment is merely an explanation of the present invention and is not intended to limit the invention. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A method for regulating castor bean inflorescence development, characterized in that: By overexpressing in castor beans PIP5K11 Genes promote early flowering in castor beans, the aforementioned PIP5K11 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. PIP5K11 The application of the gene in promoting the ripening of castor bean fruits or inhibiting the growth of primary branches in castor beans, wherein the application is the overexpression of the gene in castor beans. PIP5K11 Genes, the ones mentioned PIP5K11 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

3. PIP5K11 Application of the gene in improving castor bean pollen viability or germination rate, wherein the application is overexpression in castor beans. PIP5K11 Genes, the ones mentioned PIP5K11 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. The method according to claim 1, characterized in that, Includes the following steps: (1) Construction PIP5K11 Gene overexpression vectors were used, and the resulting recombinant plasmids were transformed into Agrobacterium competent cells using homologous recombination. (2) Using Agrobacterium-mediated transformation, the constructed [product / structure] containing [a specific ingredient / component] was [determined / contained / dissolved]. PIP5K11 The gene vector was transferred into the cotyledonary node of castor bean plant, and selection was performed. PIP5K11 Plants with overexpressed resistant genes can be domesticated and transplanted.

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