CeSUC3a gene for promoting early and multiple bulbils of taro and application thereof

Abstract

The application discloses a CeSUC3a gene for promoting early growth and multiple occurrence of taro lateral corms and application thereof, and relates to the technical field of plant genetic engineering. CeSUC3a The nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the application also provides CeSUC3a Application of the gene in cultivating taro varieties with early growth and multiple occurrence of taro lateral corms, up-regulation of the host CeSUC3a Application of the reagent for gene expression in cultivating taro varieties with early growth and multiple occurrence of taro lateral corms and the like. CeSUC3a The application overexpresses the gene in the taro with multiple taro corms by using the transgenic technology, improves the relative chlorophyll content and net photosynthetic rate of leaves in the overexpression strain, promotes early growth, early fruiting, multiple occurrence of lateral corms and multiple fruiting of taro in the overexpression strain, and further promotes early maturity and high yield of the overexpression strain, thereby providing important gene resources for early screening and identification of taro early maturity and high yield breeding materials and cultivation of early maturity and high yield taro varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to a CeSUC3a gene that promotes early and frequent growth of taro lateral corms and its application. Background Technology

[0002] taro( Colocasia esculenta (L. Schott) is a perennial herbaceous plant belonging to the genus *Colocasia* of the family Araceae. It is often cultivated as an annual. Rich in mucoproteins, polysaccharides, and other physiologically active substances, coupled with high starch digestibility and abundant vitamins and minerals, it can alleviate various sub-health symptoms, thus gaining increasing popularity among consumers. However, currently cultivated multi-branch taro varieties generally suffer from late-stage tuber formation and a limited number of tubers, which urgently need to be addressed. This severely restricts their early maturity and high yield. There is a pressing need for new early-maturing, high-yielding taro varieties that produce many tubers. Discovering and regulating genes that regulate the early and abundant formation of multi-branch taro is of great significance for breeding new early-maturing, high-yielding taro varieties and improving planting efficiency.

[0003] Sucrose transporters (SUTs / SUCs) are a class of transmembrane proteins widely found in plants. They play a key role in the long-distance transport of sucrose from source to sink and have an important impact on plant growth and development, flower fertility, and plant height. However, the function of these proteins in promoting the early and abundant development of taro lateral corms has not been reported. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a CeSUC3a gene that promotes early and frequent growth of taro lateral corms and its application.

[0005] This invention utilizes transgenic technology to overexpress in taro CeSUC3a The gene can promote the early growth of lateral corms and the early formation of taro tubers in overexpressed lines, as well as the development of more lateral corms and more taro tubers, thereby promoting early maturity and high yield in overexpressed lines. This provides important genetic resources for the early screening and identification of early-maturing and high-yielding taro breeding materials and the breeding of early-maturing and high-yielding taro varieties.

[0006] The technical solution of the present invention is as follows:

[0007] Firstly, the present invention provides a method for promoting the early and abundant growth of taro lateral corms. CeSUC3a Genes, the ones mentioned CeSUC3a The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0008] Secondly, the present invention provides the aforementioned CeSUC3a Application of genes or their biological materials in the breeding of taro varieties with early and abundant lateral corms.

[0009] This invention provides taro CeSUC3a The application of genes or related biological materials in promoting the early and abundant development of lateral corms in Amorphophallus multiflorus. Promoting the early and abundant development of lateral corms in Amorphophallus multiflorus refers to the application of genes or related biological materials in promoting the early and abundant development of lateral corms through overexpression of the aforementioned genes. CeSUC3a Genes promote early and high-yielding taro lines in transgenic multi-yielding taro strains.

[0010] In some embodiments of the present invention, the early and frequent development of lateral bulbs of the multi-seed taro includes early development of lateral bulbs of the multi-seed taro, early formation of taro tubers and frequent development of lateral bulbs, and formation of multiple taro tubers.

[0011] In some embodiments of the present invention, the biomaterial includes the... CeSUC3a A recombinant vector or recombinant bacterium expressing a gene, specifically, a recombinant vector expressing the CeSUC3a gene, the recombinant vector comprising the CeSUC3a gene, and a recombinant bacterium expressing the CeSUC3a gene, the recombinant bacterium comprising the recombinant vector.

[0012] Thirdly, this invention provides an upregulation of the host... CeSUC3a The application of gene expression reagents in breeding taro varieties with early and abundant lateral corms. CeSUC3a The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0013] Fourthly, this invention provides a method for cultivating taro varieties with early and abundant lateral corms, the method comprising adjusting the taro plant... CeSUC3a The expression of genes and / or the promotion of the production of their encoded products, said CeSUC3a The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0014] In some embodiments of the present invention, the method includes placing the CeSUC3a Genes or their biological materials are transferred into taro plants via Agrobacterium-mediated transformation, thereby increasing the concentration of these genes in the taro plants. CeSUC3a Gene overexpression. This invention will utilize the above... CeSUC3a The gene was transferred into taro plants using Agrobacterium-mediated genetic transformation technology, enabling... CeSUC3a Stable overexpression of the gene leads to early lateral bulb development, early tuber formation, and increased lateral bulb production in the overexpressing lines, resulting in early-maturing and high-yielding taro varieties.

[0015] Fifthly, the present invention provides a method for detecting [something] in taro plants. CeSUC3a A qRT-PCR primer set for gene expression level, wherein the qRT-PCR primer set includes a forward primer as shown in SEQ ID NO. 6 and a reverse primer as shown in SEQ ID NO. 7; CeSUC3a The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0016] Sixthly, the present invention provides the application of the qRT-PCR primer set described above in the identification or screening of early-maturing and high-yielding taro breeding materials.

[0017] In some embodiments of the present invention, the early-maturing and high-yielding taro breeding material includes taro with early lateral corm growth, early taro formation, and multiple lateral corms and taro formation.

[0018] This invention has at least one of the following beneficial effects:

[0019] This invention provides a method to promote the early and abundant growth of taro lateral corms. CeSUC3a Genes, and research through cloning taro CeSUC3a Genes, constructing a system containing " CaMV 35S - CeSUC3a "The overexpression vector of the fusion gene was used to study its biological function in regulating the early and frequent development of lateral corms in taro. The results showed that..." CeSUC3a The overexpression of the gene in taro lines resulted in early lateral corm formation, early fruiting of taro, and the development of multiple lateral corms and fruiting taro, thereby promoting early maturity and high yield in the overexpression lines. This provides important genetic resources for the early screening and identification of early-maturing and high-yielding taro breeding materials and the cultivation of early-maturing and high-yielding taro varieties. Attached Figure Description

[0020] Figure 1 This is a gel image of the PCR amplification product of the taro CeSUC3a gene in Example 1.

[0021] Figure 2 This is a spatiotemporal expression analysis of the CeSUC3a gene in taro in Example 3. In the figure, A represents the relative expression level of the CeSUC3a gene in different parts, and B represents the relative expression level of the CeSUC3a gene at different times.

[0022] Figure 3 For example, wild-type taro and transformed " CaMV 35S - CeSUC3a "Target gene in transgenic taro lines with fusion genes" CeSUC3a Expression analysis. **P<0.01.

[0023] Figure 4 For example, wild-type taro and transformed " CaMV 35S - CeSUC3a Phenotypic analysis of transgenic taro lines with fusion genes after 100 days of cultivation. In the figure, A represents the wild-type line (WT) and the overexpressing line. CeSUC3a The growth status of tubers in the overexpressing taro line (OE) after 100 days of cultivation; B in the figure represents the wild-type line (WT) and the overexpressing taro line. CeSUC3aThe number of tubers (n>3) in taro lines (OE) with the overexpressing gene after 100 days of cultivation; C in the figure represents wild-type lines (WT) and overexpressing genes. CeSUC3a Comparison of net photosynthetic rates of mature leaves in taro lines (OE) with the overexpressing gene (n>3); D in the figure represents wild-type lines (WT) and lines overexpressing the gene. CeSUC3a Comparison of relative chlorophyll content among taro lines (OE) of the gene (n>3). *P<0.05.

[0024] Figure 5 For example, wild-type taro and transformed " CaMV 35S - CeSUC3a Phenotypic analysis of transgenic taro lines with fusion genes after 160 days of cultivation. In the figure, A represents the wild-type line (WT) and the overexpressing line. CeSUC3a The growth status of tubers in the overexpressing taro line (OE) after 160 days of cultivation; B in the figure represents the wild-type line (WT) and the overexpressing taro line. CeSUC3a Statistical analysis of the number of daughter tubers in the overexpressing taro line (OE) (n=30); C in the figure represents the wild-type line (WT) and the overexpressing taro line. CeSUC3a Statistical analysis of the ratio of total weight of individual tubers in taro lines (OE) of the gene (n>3). *P<0.05, **P<0.01. Detailed Implementation

[0025] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] Example 1: Taro Sucrose Transporter Gene CeSUC3a Cloning

[0027] This embodiment discovered [the virus] in taro. CeSUC3a Gene, CeSUC3a The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the specific process is as follows:

[0028] (1) RNA was extracted from taro leaves using a polysaccharide and polyphenol plant RNA extraction kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., RC411-01). cDNA synthesis was performed using a reverse transcription kit (purchased from Shanghai Yisheng Biotechnology Co., Ltd., 11121ES60). First-strand cDNA was obtained by reverse transcription. For specific operating procedures, please refer to the product instructions.

[0029] (2) Design specific primer pairs for amplification. In this embodiment, the forward primer is introduced with the following: Nde I. Restriction site, introduced in the reverse primer EcoR I. Enzyme cleavage site.

[0030] Forward primer: 5'-GGAATTCCATATGATGGACGCCATCTCGATCCG-3' (introducing...) Nde I. Restriction site, SEQ ID NO. 2)

[0031] Reverse primer: 5'-GGAATTCTTAGCCAAATCCATGAAGACCCG-3' (introduced) EcoR I. Restriction site, SEQ ID NO. 3)

[0032] (3) Using the cDNA from step (1) as a template, perform PCR amplification using the above-mentioned specific primer pair to obtain CeSUC3a CDS fragments of genes.

[0033] The PCR reaction procedure is as follows:

[0034] 98℃ for 3 minutes;

[0035] 98℃ for 10 seconds, 56℃ for 20 seconds, 72℃ for 1 minute and 20 seconds, 32 cycles;

[0036] 72℃ for 5 minutes;

[0037] Keep warm at 4℃.

[0038] The PCR reaction system is shown in Table 1:

[0039] Table 1

[0040]

[0041] (4) The target DNA fragment was recovered by agarose gel electrophoresis. Figure 1 The DNA gel recovery kit (purchased from Daling Biotechnology, DLN 801) was used for recycling. For detailed operating procedures, please refer to the product manual.

[0042] (5) The recovered PCR product was subjected to DNA sequencing. Its nucleotide sequence is shown in SEQ ID NO.1. It was named as follows. CeSUC3a Gene.

[0043] Example 2: Constructing " using pRI 101-AN vector CaMV 35S - CeSUC3a "Fusion genes"

[0044] This embodiment provides a method for constructing engineered Agrobacterium for the genetic transformation of taro. The specific method is as follows:

[0045] (1) The vector pRI 101-AN plasmid was extracted from Escherichia coli and used... Nde Ⅰ / EcoRI. Large vector fragments were recovered after double enzyme digestion.

[0046] (2) The recovered product from Example 1 CeSUC3a CDS fragments of genes are used Nde Ⅰ / EcoR I. Double enzyme digestion, and recovery of the digested fragments by agarose gel electrophoresis (same as in Example 1).

[0047] (3) The two fragments were ligated overnight at 16°C using Solution I ligase to complete the ligation of the two fragments on the pRI 101-AN vector. CaMV 35S - CeSUC3a "The fusion gene was constructed to obtain the ligation product."

[0048] The connection system is shown in Table 2:

[0049] Table 2

[0050]

[0051] (4) Transform Escherichia coli DH5α competent cells using the ligation mixture, as follows:

[0052] Escherichia coli DH5α competent cells were prepared using conventional CaCl2 induction and transformation methods. The competent cells were transformed with 10 μl of ligation product and then evenly spread onto a plate containing kanamycin and incubated upside down at 37°C for 12 hours.

[0053] (5) Perform PCR reaction using plasmid as template to identify the " " in the plasmid CaMV 35S - CeSUC3a "The fusion gene amplified fragment to a size of 1833 bp. The primers used are as follows:"

[0054] Forward primer: 5'-ATGGACGCCATCTCGATCCG-3' (SEQ ID NO. 4)

[0055] Reverse primer: 5'-TTAGCCAAATCCATGAAGACCCG-3' (SEQ ID NO. 5)

[0056] (6) Extract plasmids from positive clones and transform Agrobacterium GV3101 using conventional methods to obtain engineered Agrobacterium for genetic transformation of taro.

[0057] Example 3: Obtaining transgenic taro plants

[0058] In this embodiment, two transgenic taro plants were constructed, and it was found that... CeSUC3a The expression level of the gene was significantly increased in both transgenic lines, as detailed below:

[0059] (1) The “constructed using Example 2” CaMV 35S - CeSUC3a "The fusion gene was transformed into taro callus tissue. The specific transformation method adopted was Agrobacterium-mediated callus transformation, specifically referring to Chinese Patent Publication No. CN119193684A or the reference (Gui Yanling, Zhang Yufeng, He Yining, et al. Construction of Agrobacterium-mediated taro genetic transformation system [J]. Journal of Jiangxi Agricultural University, 2024, 46 (06): 1445-1455), after 50 mg l -1 Kanamycin resistance screening was conducted. Resistant buds were cultured into seedlings, and the normally growing resistant plants were transferred to soil for culture to obtain two transgenic lines (transgenic line-1 and transgenic line-2).

[0060] (2) qRT-PCR detection of transgenic plants: After the resistant plants were transferred to soil and cultured to the four-leaf stage, total RNA was extracted from the plant tissues and reverse transcribed into cDNA according to the method in Example 1. The PCR reaction was carried out using the following primers, reaction procedure and reaction system:

[0061] CeSUC3a Primers for gene qRT-PCR detection:

[0062] Forward primer: 5'-CAGAGGACAGCCAAATTGAAGCC-3' (SEQ ID NO. 6)

[0063] Reverse primer: 5'-GCCAGCCATGTAAGTGACATCAC-3' (SEQ ID NO. 7)

[0064] Actin Primers for gene expression detection:

[0065] Forward primer: 5'-CTAGTGGTCGCACAACAGGT-3' (SEQ ID NO. 8)

[0066] Reverse primer: 5'-TTCACGCTCAGCAGTGGTAG-3' (SEQ ID NO. 9)

[0067] The reaction procedure is as follows:

[0068] 95℃ for 3 minutes;

[0069] 95℃ for 10 seconds, 60℃ for 30 seconds, 72℃ for 30 seconds, 40 cycles;

[0070] Keep warm at 4℃.

[0071] The reaction system is shown in Table 3:

[0072] Table 3

[0073]

[0074] CeSUC3a Spatiotemporal expression analysis of genes can be found in Figure 2 , Figure 2 show: CeSUC3a The gene is mainly expressed in the leaf veins, and its expression level is much higher during the day than at night. Figure 2 Further analysis of A in the text shows that... CeSUC3a Gene expression exhibits a diurnal rhythm. Figure 2 (B in the middle).

[0075] qRT-PCR results are as follows Figure 3 As shown, CeSUC3a Gene expression was significantly increased in both transgenic lines (transgenic line-1 and transgenic line-2), upregulated 8–10 times compared with the wild-type control. Figure 3 ).

[0076] Example 4: Overexpression CeSUC3a Analysis of the early and frequent occurrence of lateral corm traits in taro lines

[0077] This embodiment analyzed the early and frequent occurrence of lateral corms in taro lines overexpressing the CeSUC3a gene. The specific method is as follows:

[0078] (1) Analysis of the early and frequent lateral bulb development trait in transgenic lines after 100 days of planting:

[0079] The transgenic lines identified by qRT-PCR in Example 3 were planted for 100 days to detect the early and multiple lateral bulb formation traits. Specific method: Wild-type and overexpressing transgenic lines were... CeSUC3a The transgenic taro lines were planted in a greenhouse (transplanted on February 21, 2025) under standard water and fertilizer management. The net photosynthetic rate and relative chlorophyll content of mature leaves were measured. Harvesting took place on the 100th day (June 1, 2025), and the tuber formation was observed, with the number of daughter tubers counted. The experimental results are as follows: Figure 4 As shown, overexpression CeSUC3a Compared to wild-type taro lines, the daughter tubers of the genetically modified taro lines swell earlier, form tubers earlier, and produce more daughter tubers. Figure 4 A and Figure 4 In type B), the net photosynthetic rate and relative chlorophyll content of the leaves were significantly higher than those of the wild type. Figure 4 C and Figure 4 (D in the middle).

[0080] (2) Analysis of the early and frequent lateral bulb development trait in transgenic lines after 160 days of planting:

[0081] The transgenic lines identified by qRT-PCR in Example 3 were planted for 160 days to detect the early and multiple lateral bulb formation traits. Specific method: Wild-type and overexpressing transgenic lines were... CeSUC3a The taro lines were grown in a greenhouse (transplanted on February 21, 2025) under standard water and fertilizer management. Harvesting took place after 160 days (July 31, 2025), and the tuber formation characteristics were observed. The number and weight of the daughter tubers were recorded. The experimental results are as follows: Figure 5 As shown, compared to wild-type strains, overexpression CeSUC3a The number of daughter tubers in the taro lines of the gene was significantly increased. Figure 5 A and Figure 5 In section B), the average total weight per plant also increased significantly. Figure 5 (C in the middle).

[0082] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Upregulate the host CeSUC3a Gene expression or CeSUC3a The application of genetic biomaterials in the breeding of taro varieties with early and abundant lateral corms is characterized by, The CeSUC3a The nucleotide sequence of the gene is shown in SEQ ID NO. 1, and the biological material comprises the gene. CeSUC3a Gene recombinant vectors or recombinant bacteria.

2. The application according to claim 1, characterized in that, The term "early growth and multiple development of lateral bulbs" refers to the early growth of lateral bulbs, early formation of lateral bulbs, and multiple lateral bulb formation.

3. A method for cultivating taro varieties with early and abundant lateral corms, characterized in that, The method includes: adjusting the taro plant... CeSUC3a The expression of genes and / or the promotion of the production of their encoded products, said CeSUC3a The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

4. The method according to claim 3, characterized in that, The method includes: placing the... CeSUC3a Genes or their biological materials are transferred into taro plants via Agrobacterium-mediated transformation, thereby increasing the concentration of these genes in the taro plants. CeSUC3a Gene overexpression.

5. Application of qRT-PCR primer sets in the identification or screening of early-maturing, high-yielding taro breeding materials, characterized in that... The qRT-PCR primer set includes a forward primer as shown in SEQ ID NO. 6 and a reverse primer as shown in SEQ ID NO.

7.

6. The application according to claim 5, characterized in that, The early-maturing and high-yielding taro breeding materials include those with early-growing lateral bulbs, early-forming taro tubers, and multiple lateral bulbs that produce many taro tubers.

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