CN117987424A - Application of tea CsAPL gene in regulating and controlling plant flowering - Google Patents

Abstract

The invention provides an application of a tea CsAPL gene in regulating and controlling plant flowering, belonging to the technical field of agricultural biology. The agrobacterium-mediated flower dipping method is adopted to transform CsAPL.sup.1 over-expression vector into wild type Arabidopsis thaliana, and the obtained transgenic Arabidopsis thaliana has flowering and fruiting time obviously longer than that of the wild type Arabidopsis thaliana. The fluorescent quantitative qRT-PCR detection shows that the expression quantity of CsAPL gene in the bud differentiation and flower organ development period of the multi-flower variety of tea tree is obviously higher than that of the few (no) flower variety. The plant expression vector containing CsAPL gene is applied to tea tree, can accelerate the flowering and fruiting of tea tree, shorten the breeding period, or apply CsAPL gene to early breeding identification of new variety with few (no) flowers of tea tree, and has excellent application value.

Description

Application of tea CsAPL gene in regulating and controlling plant flowering

Technical Field

The invention belongs to the technical field of agricultural biology, and particularly relates to application of a tea CsAPL gene in regulating and controlling plant flowering.

Background

Tea tree [ CAMELLIA SINENSIS (L.) O.Kuntze ] belongs to tea seed plants of camellia genus of Theaceae, and is an important leaf economic crop in China. Tea is deeply favored by consumers due to the nutritional value and health efficacy of the tea, and the tea industry plays an important role in helping farmers to get rid of poverty and become rich and in the rural industry to reach the best. The tea tree has the characteristics of multiple flowers and long flowering period, and the period from flower bud differentiation to fruit maturation is 15-16 months. In agricultural production, the long and vigorous reproductive growth of tea trees consumes a large amount of nutrients, and has adverse nutrition competition effect on the growth of young sprout leaves, so that the yield and quality of tea leaves are affected, and finally the improvement of the economic benefit of tea industry is limited. At present, a transgenic technology system of tea trees is not established, and sexual cross breeding is still one of main methods of tea tree breeding, and has the defects of long flowering and fruiting period, low fruiting efficiency and the like. Therefore, the molecular regulation mechanism of the flower formation of the tea tree is clarified, and the method is of great significance in guiding the selection and breeding of new varieties with early flowers or few (no) flowers, shortening the breeding period of the tea tree or promoting the nutrition growth of the tea tree.

The flowering of plants is a complex process determined by self genetic materials and external environmental factors (light length, light quality, temperature and the like), is a physiological phenomenon regulated and controlled by specific expression of a series of flowering genes in time and space, and has important roles of reproducing offspring and adapting to ecological environment. The gene coding protein of tea CsAPL has MADS and K-box conserved domains, and belongs to members of the AP1/FUL subfamily (also called AP1-like and SQUA subfamilies) of the MADS-box family. AP1 (APETALA 1) is a typical floral meristem class A gene involved in floral transformation, floral development and floral organogenesis in plants. Studies in model plants indicate that mutation of the AP1 gene results in significant changes in the Arabidopsis flower type, as well as affected sepals, petal morphology, number and locus; AP1 and LEAFY interact to regulate expression of downstream flowering-time genes SVP, SOC1, and the like. At present, the research on the flower formation mechanism of tea trees is not perfect, and the gene function of tea CsAPL1 is not reported. Therefore, the physiological function of the CsAPL gene in the flowering process of plants and the correlation between the physiological function and the flowering of the tea trees are analyzed and clarified by over-expressing CsAPL genes in arabidopsis and detecting the expression quantity of CsAPL1 in tea tree varieties with different flowering characteristics by using fluorescent quantitative qRT-PCR.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to design a technical scheme for providing the application of the tea CsAPL gene in regulating and controlling the flowering of plants.

The invention is realized by the following technical scheme:

in one aspect, the invention provides application of the tea CsAPL gene in regulating and controlling plant flowering.

Further, the nucleotide sequence of the CsAPL gene is shown as SEQ ID NO. 1.

Further, the amino acid sequence of the CsAPL gene coding protein is shown as SEQ ID NO. 2.

Further, regulating plant flowering is specifically to accelerate plant flowering and fruiting.

Further, the plants include woody plant tea tree and herb plant Arabidopsis thaliana.

Further, the application method of the invention specifically comprises the following steps: 1) Allowing the plant to contain CsAPL genes; or 2) over-expressing CsAPL genes in plants.

Further, the mode 2) adopts an agrobacterium-mediated method to transfer a recombinant vector containing the tea CsAPL gene into a plant genome, and the transgenic plant with CsAPL1 over-expression is obtained by screening.

Further, the carrier is pCAMBIAS1300,1300.

In another aspect, the present invention provides a method of regulating flowering in a plant comprising the steps of: 1) Allowing the plant to contain CsAPL genes; or 2) over-expressing CsAPL genes in plants.

In another aspect, the invention provides the use of the tea CsAPL gene in the assisted identification of multiple, fewer or no flower varieties of camellia sinensis.

The invention separates 1 CsAPL genes closely related to the differentiation and development of camellia from tea tree tissues, and researches find that the genes are positioned in cell nuclei. The fluorescence quantitative qRT-PCR proves that CsAPL gene has high expression under the conditions of flower bud tissue and long sunlight, and the CsAPL1 is supposed to play a key role in early flower bud development. The molecular technology is used to construct CsAPL gene over-expression carrier, which is transferred into wild Arabidopsis to be over-expressed, so that the flowering and fruiting time of Arabidopsis can be shortened. The invention provides a favorable gene resource for breeding new varieties of tea trees with different flower formation characteristics.

Drawings

FIG. 1 is an agarose gel electrophoresis of PCR amplification products of tea CsAPL gene.

FIG. 2 is a phylogenetic tree analysis of the MADS-box family proteins of tea tree and Arabidopsis thaliana.

FIG. 3 is an amino acid sequence alignment of CsAPL1 with a member of the tea tree, arabidopsis AP1-like subfamily.

FIG. 4 shows subcellular sites of tea CsAPL protein. GFP: a green fluorescent signal; RFP: a nuclear red fluorescent signal; light: a bright field; merged: and (5) signal fusion.

FIG. 5 is a tissue-specific expression analysis of tea CsAPL gene. A: spring 'Longjing 43' different tissue organs; b: autumn 'Longjing 43' different tissue organs; c: the full bloom stage 'Longjing 43' is different flower organs.

FIG. 6 shows the amount of CsAPL gene expressed at different photoperiod in a room. LD:16h of illumination/8 h of darkness, long sunlight; SD:8h light/16 h dark, short day.

FIG. 7 is a graph depicting the flowering phenotype characteristics of Arabidopsis thaliana overexpressed by tea plant CsAPL 1. A: expression level of CsAPL in wild-type and overexpressed Arabidopsis; b: 20d phenotype under long-day LD conditions; c, 40d phenotype under short-day SD conditions; d: 55d phenotype under short-day SD conditions.

FIG. 8 shows the flowering characteristics in the field for different tea varieties. A: zhuang No. 2 (ZH 2) and Fuding Dabaicha' (FDDB) of the multi-flower variety; b: few (none) flower varieties 'Pingyang super early' (PYTZ) and 'red bud fingered citron' (HYFS).

FIG. 9 is an axillary bud/flower bud tissue observation of multiple and few (none) flower varieties of tea tree. FM: flower meristem; white line scale: 0.5cm; ZH2: 'Zhonghuang No. 2'; FDDB: 'Fuding Dabai tea'; PYTZ: 'Pingyang te early'; HYFS: 'Hongya bergamot'.

FIG. 10 shows the expression level of the tea CsAPL gene in a variety of flower-rich/flower-less (no) flower tea. A: axillary buds/flower buds; b: mature leaves; ZH2: 'Zhonghuang No. 2'; FDDB: 'Fuding Dabai tea'; PYTZ: 'Pingyang te early'; HYFS: 'Hongya bergamot'.

Detailed Description

The invention is further illustrated below in connection with examples, which are not intended to limit the scope of the invention. Unless otherwise indicated, reagents, consumables and the like used in the examples are commercially available, and the examples are carried out according to conventional experimental methods (Wei Chao, tea plant biological experimental techniques, 2023) or according to the manufacturer's instructions.

Example 1: cloning and sequence analysis of tea CsAPL1 Gene

Based on the existing tea tree transcriptome and genome database of the subject group, 1 gene CsAPL1 encoding MADS and K-box conserved protein domains was retrieved. The upstream and downstream specific primers F and R were designed, and the target fragment of CsAPL gene was obtained by PCR and KOD-plus-Neo high-fidelity enzyme (Toyobo, japan) using the tea tree 'Longjing 43' axillary bud/leaf cDNA as a template (FIG. 1). The target fragment is recovered and purified and then connected to a Blunt-zero vector (Transgen, china), the recombinant vector is transformed into T ₁ competent escherichia coli (Transgen, china), and positive monoclonal bacterial liquid is selected and sent to Hangzhou Kangshen biotechnology Co Ltd for sequencing. As a result, the complete open reading frame ORF sequence (693 bp) of CsAPL gene was obtained, the nucleotide sequence of which is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein of which is shown as SEQ ID NO. 2. The upstream primer F used: 5'-ACAAGAGAGAAAATGGGGAGAGGA-3' (SEQ ID NO. 3); the downstream primer R:5'-CATGCCTTGAAATTTATCCATTGATG-3' (SEQ ID NO. 4).

Protein sequences of 83 tea plant MADS-box transcription factors and 76 Arabidopsis thaliana MADS-box transcription factors were collected from TPIA (Tea Plant Information Archive) and TAIR (The Arabidopsis Information Resource) databases, and the CsAPL1 protein sequence was subjected to phylogenetic tree and multiple sequence alignment analysis with Arabidopsis thaliana, tea plant MADS-box family proteins using MEGA 7.0 software. The results of the evolutionary tree showed that CsAPL1 was most closely related to TEA tree TEA030609.1, TEA007284.1, and Arabidopsis AT5G60910.1 (AGL 8/FUL), AT1G26310.1 (AGL 10/CAL), AT1G69120.1 (AGL 7/AP 1), AT3G30260.1 (AGL 79), all belonging to the AP1-like subfamily members (FIG. 2). Amino acid sequence alignment showed that CsAPL was a cloned new TEA transcript (FIG. 3) with multiple amino acid sites different from TEA030609.1, TEA007284.1, AT5G60910.1, AT1G26310.1, AT1G69120.1, AT3G30260.1.

Example 2: subcellular localization of tea CsAPL protein

Plant subcellular localization vector 35S sGFP was selected for double cleavage at KpnI and SalI sites, the cleavage system was referred to FASTDIGEST (Thermofisher, USA) and reacted at 37℃for 3h, and the cleavage products were subjected to 1.0% agarose gel electrophoresis and purified for vector backbone using AxyPrep DNA gel recovery kit (Axygen, USA). PCR amplification primers, SL-F, were designed according to the ORF sequence of CsAPL gene and DNA seamless cloning specifications: 5'-GTCGGAGCTCGGTACCATGGGGAGAGGAAGAGTCCAG-3' (shown in SEQ ID NO. 5) and SL-R:5'-TGCTCACCATGTCGACTCCATTGATGTGACTAATCATCCA-3' (SEQ ID NO. 6). The target DNA fragment was amplified by KOD-plus-Neo high-fidelity enzyme (Toyobo, japan) and PCR reaction using the correctly sequenced Blunt-zero-CsAPL1 vector as a template. The DNA insert and the linearization vector are subjected to recombination connection reaction according to a seamless cloning instruction (Vazyme, china), the recombination product is transformed into T ₁ competent escherichia coli (Transgen, china), and after bacterial liquid PCR is used for verifying positive monoclonal bacterial liquid, the positive monoclonal bacterial liquid is sent to Hangzhou Kangshen Biotechnology Co., ltd for sequencing, and finally the correct 35S CsAPL S sGFP vector is obtained.

35S CsAPL S sGFP recombinant vector and 35S sGFP empty vector are transformed into competent cells of agrobacterium GV3101 by a freeze thawing method, bacterial liquid is coated on a screening culture medium of LB+kanamycin (Kan) +rifampicin (Rif), and the culture medium is subjected to dark culture at 28 ℃ for 48 hours, and monoclonal colonies are selected for PCR identification. Agrobacterium carrying 35s:: csAPL:: sGFP was inoculated into 20mL of liquid medium LB+Kan+Rif, shaken at 28℃at 200rpm until OD ₆₀₀ =1.0, centrifuged at 5000rmp for 10min, and the supernatant was discarded, and the cells were resuspended with an appropriate amount of counterstain (10 mmol L ^-1MES,10mmol L^-1MgCl₂,150μmol L^-1 acetosyringone) to give OD ₆₀₀ =0.8. The suspension was injected onto the back of 4-week-old tobacco leaves using a sterile syringe, and after 2d of normal culture, it was observed with a laser confocal microscope (Zeiss, germany) and photographed. The results showed that CsAPL a fusion GFP green fluorescent signal could overlap with the nuclear RFP red fluorescent signal, presenting a yellow signal (fig. 4). It was shown that tea CsAPL protein localizes to the nucleus.

Example 3: gene expression analysis of tea CsAPL1

In a natural field test field (30.18°n,120.09 °e) of tea tree 'Longjing 43', top buds, first leaves, second leaves, third leaves, mature leaves, stems and root tissues are picked in spring (4 months of 2020), top buds, axillary buds, flower buds, flowers, mature leaves, stems, roots and fruit tissues are picked in autumn (10 months of 2020), and flower buds, petals, stamens, pistil, calyx and flower stalk tissues are picked in a full bloom stage (11 months of 2021). The healthy 'Longjing 43' potted seedlings with consistent growth vigor are moved into a climatic chamber (16 h light/8 h dark at 25 ℃ and 8h light/16 h dark) for 15d in 20 days of 2022, sampled at the first time point (two leaves of one bud) at 5:00a.m. on 4 days of 8 months, and sampled every 4 hours in 48 hours.

Total RNA and cDNA were obtained using the polysaccharide polyphenol plant total RNA extraction kit (Tiangen, china) and the PRIMESCRIPT RT REAGENT reverse transcription kit (Takara, china). Tea tree CsPTB (NCBI accession number: GAAC 01052498.1) was selected as an internal reference gene, and primers were q-CspTB.F:5'-ACCAAGCACACTCCACACTATCG-3' (shown in SEQ ID NO. 7) and q-CsPTB.R:5'-TGCCCCCTTATCATCATCCACAA-3' (SEQ ID NO. 8). Fluorescent quantitative primers for CsAPL gene, q-CsAPL1.F, were designed by NCBI Primer-BLAST program: 5'-GAGCTTCAAAAAAAGATTAAGGAAAAGG-3' (shown in SEQ ID NO. 9) and q-CsAPL1.R:5'-GGCATTACTGCATTGTTTTGAATTTGAT-3' (SEQ ID NO. 10). Tissue-specific expression patterns and photoperiod response expression levels of the CsAPL gene were detected using the LightCycler 480SYBR Green I Maste reagent and the LightCycler 480 II instrument (Roche, switzerland).

In the spring 'Longjing 43' different tissues, csAPL1 was expressed in the highest amount in the mature leaves, followed by relatively higher amounts in the root, second leaf and third leaf, and relatively lower amounts in the terminal bud, first leaf and stem (FIG. 5A). In different tissues of the 'Longjing 43', csAPL1 had the highest expression level in flower buds, higher expression level in terminal buds and axillary buds, lower expression level in flower buds, flowers, mature leaves and roots, and lowest expression level in stems and fruits (FIG. 5B). By using different flower organs in flowers of 'Longjing tea 43' as materials, through detection, csAPL1 has the highest expression level in flower buds and has lower expression level in flower buds, flowers and petals, stamens, pistil, calyx and pedicel (figure 5C). The results indicate that CsAPL1 plays a major role in early flower bud development.

Under both long and short day conditions, csAPL gene expression did not respond to circadian rhythms without significant changes. However, transcript abundance of CsAPL gene was consistently higher under long-day conditions than under short-day conditions (FIG. 6). It was demonstrated that CsAPL gene increased expression in response to long-term sunlight.

Example 4 functional analysis of tea tree CsAPL1 Gene

Plant over-expression vector pCAMBIAS1300 selects ApaI and SalI sites for double enzyme digestion, and designs a vector construction primer OE-F:5'-GCTTCTGCAGGGGCCCATGGGGAGAGGAAGAGTCCAG-3' (SEQ ID NO. 11) and OE-R:5'-CTCCCATATGGTCGACTTATCCATTGATGTGACTAATCATCCA-3' (SEQ ID NO. 12). Vector double enzyme digestion, DNA target fragment amplification, recombinant ligation reaction, transformed E.coli, recombinant vector sequencing, and transformed Agrobacterium were performed as described in reference example 2. 1mL of agrobacterium containing OE-CsAPL1 vector was inoculated into 200mL of LB+Kan+Rif liquid medium, cultured at 28℃at 200rpm until OD ₆₀₀ =1.0, the bacterial solution was centrifuged at 5000rpm for 10min, and the supernatant was discarded; re-suspending the thallus with 200mL of infection liquid (1/2MS+5% sucrose, pH=5.7), adding 0.03% Sliwet L-77 surfactant, and stirring with glass rod; selecting wild arabidopsis thaliana in a bolting period, cutting off white flower buds and pods, placing inflorescences in an invasion solution for soaking for 2min, taking out the arabidopsis thaliana, performing dark culture for 24h, and then recovering normal culture until T ₀ -generation seeds are harvested; and (3) carrying out T ₁ generation positive seedlings and T ₂ generation single copy genome insertion screening on the Arabidopsis seeds by using a 1/2MS culture medium containing hygromycin HYG, and finally propagating the seeds to obtain T ₃ generation homozygous seeds.

Arabidopsis seeds (OE-1, OE-2 and OE-3, FIG. 7A) with different amounts of CsAPL.sup.1 overexpression and wild-type Col-0 were sown in nursery blocks and placed at 22℃under 16h light/8 h dark (LD) and 8h light/16 h dark (SD) conditions for phenotypic observation. Under long-day conditions, after 20d sowing, the over-expression strains OE-2 and OE-3 are found to have obvious bolting and flower buds and have a phenotype of flowering in advance compared with the wild Col-0 (figure 7B); under short-day conditions, over-expressed lines OE-2 and OE-3 began bolting and sprouting after sowing for 40D (FIG. 7C) and 3 over-expressed lines had all been flowering after sowing for 55D, with OE-2 and OE-3 lines having had pods grown (FIG. 7D) as compared to wild-type Col-0. The result shows that the flowering of the transgenic arabidopsis with higher CsAPL-1 over-expression quantity is remarkably advanced under long-day or short-day conditions.

Example 5 evaluation of tea CsAPL Gene for use in tea tree varieties with different flowering Properties

In 2022 autumn, the field flowering traits of 4 tea tree varieties 'Zhuang No. 2', 'Fuding Dabai tea', 'Pingyang Te early', 'Hongyangzhu' in the natural growth test lands (30.18 ℃ N,120.09 ℃ E) were observed. From the flowering phenotype of the 4 varieties in the field, the 'Zhonghuang No. 2' and the 'Fuding big white tea' are the blooming tea varieties, and flower buds, flower buds and flowers are hung on branches of the tea varieties (figure 8A); whereas 'Pingyang terzao' and 'Hongyao fingered citron' are tea tree varieties with little flowering, little flower buds are observed on the branches, and only a few sporadic flowers are at the two ends of the tree row (fig. 8B). Thus, there is a significant difference in flowering number between tea tree varieties with more flowers and few (none) flowers. 'Zhonghuang No. 2' and 'Fuding big white tea' are varieties of multi-flower tea trees, but the flowering characteristics of the two varieties are different, wherein the quantity of the axillary buds of the 'Zhonghuang No. 2' leaves is large, the flowers are small and dense, the flowering period is durable, the flowering is very luxuriant, the quantity of the axillary buds of the 'Fuding big white tea' leaves is less than that of the 'Zhonghuang No. 2', but the flowers are larger, and the flowering is vigorous.

The tea flower is complete flower or amphoteric flower. In general, flower buds of tea tree differentiate from 6 months each year, and then every month. During the period of 5 months of 2022-10 months of 2022, the axillary buds/flower buds and mature leaf tissues of 4 tea tree varieties 'Huang No. 2', 'Fuding Dabaicha', 'Pingyang Tezao' and 'Hongyang fingered citron' which naturally grow in a test place are collected, the morphological characteristics of the axillary buds/flower buds are observed, and a photo record is made. As can be seen from the photographed pictures of axillary buds/flower buds (fig. 9): at the 29 th month of 2022, the axillary buds of the 'Zhonghuang No. 2' grow into macroscopic flower bud tissues, which means that the 'Zhonghuang No. 2' has flower meristems (Floral meristem, FM) at the moment, and then FM is continuously differentiated to form sepals, petals, stamens and pistil primordium, enter the development of flower organs, and the flower buds show a continuously-elongated and expanded growth state; the FM tissue can be obviously seen on day 18 7 of 2022 after the axillary leaf bud edge of the Fuding big white tea grows out, the time is later than the time of 'Zhonghuang No. 2', and then flower bud differentiation and flower organ development are started, and the flower buds also show continuous elongation and expansion. However, during days 5, 2022, 16 to 10, 13, the few (none) flower varieties 'Pingyang terzao' and 'red bud fingered citron' only see the growth of axillary buds becoming large, no formation of FM tissue was observed, and failed flower bud tissue transformation may be responsible for the few (none) flower phenotype of these 2 varieties.

The fluorescent quantitative qRT-PCR result shows that the expression level of CsAPL gene in axillary buds/flower bud tissues of 'Zhonghuang No. 2' and 'Fuding Dabai tea' is obviously higher than that of 'Pingyang terzao' and 'red bud fingered citron' in the period of 14 days to 10 months 13 days of 2022 (flower bud differentiation to flower organ development), and the gene expression is continuously up-regulated along with the development of the flower bud; csAPL1 Gene has higher expression level in early axillary buds of 'Pingyang super early' and 'Hongyao fingered citron', csAPL expression continuously decreases and maintains low expression level along with the growth and development of axillary buds (figure 10A). The CsAPL gene showed no significant difference in expression level in the mature leaf tissue of each variety, and had similar expression change trend (fig. 10B). The result shows that CsAPL has positive correlation between the expression level of the CsAPL in the axillary buds/flower buds and the flowering quantity of the tea tree, and can be used for assisting in early identification of new varieties of more flowers/less (no) flower tea trees.

While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Claims (10)

1. The application of the tea CsAPL gene in regulating and controlling plant flowering.

2. The use according to claim 1, characterized in that: the nucleotide sequence of CsAPL gene is shown as SEQ ID NO. 1.

3. The use according to claim 1, characterized in that: the amino acid sequence of CsAPL gene coded protein is shown as SEQ ID NO. 2.

4. A use according to any one of claims 1-3, characterized in that: regulating plant flowering is specifically to accelerate plant flowering and fruiting.

5. The use according to claim 4, characterized in that: the plants include woody plant tea tree and herb plant Arabidopsis thaliana.

6. A use according to any one of claims 1-3, characterized by comprising the following means: 1) Allowing the plant to contain CsAPL genes; or 2) over-expressing CsAPL genes in plants.

7. The use according to claim 6, characterized in that: the mode 2) adopts an agrobacterium-mediated method, a recombinant vector containing a tea CsAPL gene is transferred into a plant genome, and a CsAPL1 over-expressed transgenic plant is obtained through screening.

8. The use according to claim 7, characterized in that: the carrier is pCAMBIAS1300,1300.

9. A method of controlling flowering in a plant comprising the steps of: 1) Allowing the plant to contain CsAPL genes; or 2) over-expressing CsAPL genes in plants.

10. The application of the tea CsAPL gene in auxiliary identification of varieties of flowers, flowers or no flower tea trees.