NL2033935B1

NL2033935B1 - Tea plant aquaporin gene csaqp95 and application thereof

Info

Publication number: NL2033935B1
Application number: NL2033935A
Authority: NL
Inventors: Zhang Zhaoliang; Luo Xiaocao; Lian Xiaohong; Yang Tianyuan; Fan Xiaojuan; Wang Junjie; Zhao Xinpeng
Original assignee: Univ Anhui Agricultural
Priority date: 2022-04-29
Filing date: 2023-01-10
Publication date: 2023-11-13
Also published as: CN114853859B; CN114853859A

Abstract

Provided is a tea plant aguaporin gene CSAQP95 and an application thereof. The tea plant aguaporin. gene CSAQP95 has a nucleotide sequence shown in SEQ ID NO: l in the sequence listing; a protein coded by the tea plant aguaporin gene CSAQP95 has an amino acid 5 sequence shown in SEQ ID NO: 2 in the sequence listing. Expression of the tea plant aguaporin gene CSAQP95 promotes plant growth and accumulation of plant biomass. The CSAQP95 is highly expressed in terminal buds, tender leaves, and roots of tea plants. A pTCK303— CsAQP95 plasmid constructed by the gene is transformed into wild 10 type (WT) and urea uptake deficient type Arabidopsis thaliana mutant atdur3, and the biomass and yield of A. thaliana are significantly increased. Cloning and application of the gene help promote a genetic improvement process aiming at increasing the tea yield, and facilitate green, sound and sustainable development of 15 the tea industry. (+ Fig. 3)

Description

TEA PLANT AQUAPORIN GENE CSAQPZ5 AND APPLICATION THEREOF

TECHNICAL FIELD

The present disclosure relates to the technical field of ge- netic engineering, in particular to a tea plant aquaporin gene

CsAgP95 and an application thereof.

BACKGROUND

Tea plant (Camellia sinensis (L.) O. Kuntze) is an important cash crop whose leaf can be commercially used, and long-term pick- ing of new buds will take away substantial nitrogen. In tea plan- tation, urea is usually used as the main nitrogen source for tea plants. However, excessive use of urea can cause soil acidifica- tion, soil hardening, water eutrophication, and other ecological and environmental problems. Studies have shown that aquaporin (AQP) plays an important role in the efficient absorption and uti- lization of urea by plants. Therefore, investigating the physio- logical functions of the aquaporin gene in the roots of tea plants will help reveal the molecular mechanism of urea absorption by, help improve the nitrogen absorption and utilization efficiency of tea plants, and provide theoretical basis and functional gene re- sources for cultivating new tea cultivars with efficient nitrogen utilization in the tea plant.

SUMMARY

An objective of the present disclosure is to provide a tea plant aquaporin gene CsAQP95 and an application thereof, enriching the research on aquaporin in tea plant, providing a new idea for improving biomass of tea plant, and providing theoretical and practical reference basis for realizing increased accumulation of biomass of tea plants.

To achieve the above objective, the present disclosure pro- vides the following technical solutions:

In a first aspect of the present disclosure, a tea plant aq- uaporin gene CsAQP95 is provided, where the tea plant aquaporin gene CsAQP95 has a nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

Further, the present disclosure further provides a protein sequence encoded by the tea plant aquaporin gene CsAQP95, where the protein sequence is shown in SEQ ID NO: 2 in the sequence listing.

In another aspect of the present disclosure, a tea plant ex- pression vector pTCK303-CsAQP95 is provided, where the expression vector is obtained by enzyme digestion of a fragment shown in SEQ

ID NO: 1 to a pTCK303 vector.

In still another aspect of the present disclosure, use of a tea plant aquaporin gene CsAgP95 in increasing plant biomass is provided.

In yet another aspect of the present disclosure, a method for increasing plant biomass using a tea plant aquaporin gene CsAQP95, including the following steps: step 1, preparing a transformation solution: transforming a

PTCK303-CsAQP95 vector into EHA105 Agrobacterium Competent cells by a freeze-thaw method, and identifying a positive clone by con- ventional PCR assay to prepare the transformation solution; and step 2, soaking a plant inflorescence in the transformation solution, letting stand in the dark for 24 h for normal culture, harvesting seeds, placing harvested seeds in a centrifuge tube, sterilizing, drawing the seeds on MS agar by using a pipette tip, vernizing the seeds in the dark at 4°C for 72 h, transferring the seeds to a culture chamber at 23°C; culturing the seeds under a 16 h light/8 h dark cycle for two weeks, selecting and transplanting resistant plants with green leaves and normal root development in- to a cultivation substrate for further cultivation, allowing the cultivation substrate to fully absorb water before transplanting, covering a preservative film after transplanting, and removing the preservative film for culture after three days, where biomass of

T2 seeds obtained is improved.

Further, in step 1, the transformation solution is specifi- cally prepared according to the following steps: picking a posi- tive colony comprising a target gene, and culturing the positive colony in an LB broth supplemented with corresponding antibiotics at 28°C for 24 h; pipetting a cultured bacterial suspension into a fresh LB broth supplemented with corresponding antibiotics, and further conducting shake culture until ODgy is 1.0; centrifugally collecting cells, resuspending the cells in a 5wt3 sucrose solu- tion until the ODgy is 0.8; and shaking the cells well with a 0.1wt% organosilicon surfactant to obtain the transformation solu- tien.

Further, in step 2, a sterilization method is as follows: sterilizing the seeds with 75% (v/v) ethanol for 1 min and with 10wt% NaClO; for 5 min, and rinsing the seeds with sterile water 5- 6 times.

Compared with the prior art, the present disclosure has the following beneficial effects:

In the present disclosure, the tea plant aquaporin gene

CsAQgP95 is cloned and verified for the first time, and the gene can significantly improve plant height and plant biomass. The pre- sent disclosure further provides a recombinant plasmid containing the CsAQP95 gene and a transgenic engineering strain (namely, an engineering strain obtained by transforming the pTCK303-CsA9P95 vector into EHA105 Agrobacterium Competent cells). The present disclosure enriches the research on the physiological function of the tea plant aquaporin gene, and provides genetic resources and theoretical basis for cultivating new tea cultivars with efficient nitrogen nutrient utilization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates expression patterns of a tea plant aqua- porin gene CsAQP95 in different tissues in the example of the pre- sent disclosure;

FIG. 2 illustrates the response of a tea plant aquaporin gene

CsAQP95 to different forms of nitrogen treatments in the example of the present disclosure;

FIG. 3 illustrates the subcellular localization of a tea plant aquaporin CsAQP95 in the example of the present disclosure; and in FIG. 4, A illustrates phenotypes of wild-type, CsAQP95 overexpressed, and CsAQP95 complemented mutant materials; B illus-

trates seeds of individual plants of the wild-type, CsAQP95 over- expressed, and CsAQP95 complemented mutant materials; C illus- trates plant heights of the wild-type, CsAQP95 overexpressed, and

CsAQP95 complemented mutant materials; D illustrates the biomass of the wild-type, CsAQP35 overexpressed, and CsAQP95 complemented mutant materials; E illustrates the seed weight per plant of the wild-type, CsAQP95 overexpressed, and CsAQP95 complemented mutant materials.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Technical solutions in the example of the present disclosure will be clearly and completely described below with reference to the accompanying drawings in the example of the present disclo- sure. Apparently, the described example is only a part of but not all of the examples of the present disclosure. Based on the exam- ple of the present disclosure, all other examples obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present disclosure. 1. Cloning and sequence structure analysis of the CsAQP95 gene

The cloning and sequence structure analysis of the tea plant

CsAQP95 gene were as follows:

The tea plant cultivar C. sinensis 'Shuchazao' was planted in

Nongcuiyuan, Anhui Agricultural University, and tender roots were used for RNA extraction. Total RNA extraction was carried out ac- cording to the instructions and operations of the RNA Kit (Tiangen, Beijing, China), and the RNA content and quality were detected using a spectrophotometer.

The first strand was generated by reverse transcription: with 1 ug of RNA as a template, a reaction buffer was prepared accord- ing to the instructions of PrimeScript II lst Strand cDNA Synthe- sis Kit (Takara Biotech, China), where 0.6 pL of Oligo dT Primer (50 pM), 0.4 pL of Random 6mers (50 pM), and 1 pL of dNTP Mixture (10 mM each) were added, and the reaction system was made up to 10 uL with RNase Free ddH.0; the RNA was denatured at 65°C for 5 min and immediately placed on ice. Subsequently, the above reaction buffer was added with 4 pL of 5x PrimerScript Buffer, 0.5 pL of

RNase Inhibitor (40 U}, and 1 uL of PrimerScript RTase (200 U) made up to 20 pL with ddH.0, and incubated at 42°C for 45 min, and reverse transcriptase was inactivated at 95°C for 5 min. After op- timization, a quantity of reverse transcription product was taken 5 for subsequent PCR. The CsAQP95 gene was amplified by conventional

PCR using the first-strand cDNA as an RT-PCR template. The up- stream primer was 5'-GGGGTACC ATGCCGATGATCTACGTGG-3', and the downstream primer was 5'-GGACTAGTTTAGTAATCAGCGGTGGGC-3'. The 20 pL

PCR system was: 2.5 UL of 10xEx tag buffer, 2.0 pL of dNTP, 1 ul each of upstream and downstream primers, 0.2 |L of Ex tag, 1 pL of template, and 15.8 pL of ddH;O.

The reaction program was as follows: initial denaturation at 98°C for 10 s, 35 cycles of denaturation at 98°C for 10 s, anneal- ing at 57°C for 30 s, and extension at 72°C for 2 min; and exten- sion at 72°C for 10 min. The PCR product CsAQP95 gene was puri- fied, recovered, and ligated to the pGEM-T Easy Vector (Promega,

Shanghai, China) to obtain a pGEM-T Easy::CsAQP95 plasmid, which was transformed into Escherichia coli DH5a Competent Cells and sent to GM for sequencing. The nucleotide sequence of the CsAQPI5 gene obtained is shown in SEQ ID NO: 1 in the sequence listing, which was specifically shown as follows:

ATGCCGATGATCTACGTGGATCGGATTACTCGCCGGATCGCGSTCGGAAACCGGGAA-

GAGGCGACCCACCCCGCCGCTCTCAAGGCGGCGCTGGCGGAG-

TTCATCTCAACCCTAATTTTCGTCTTCGCGGGCCAGGGATCCGGGATGGCCTTCAATAA-

GATCACTCATAGCAGCTTCACTACCCCCTCCGGCCTCATCGCCGCCGCTATTGCCCAC-

GCATTCGGACTTTTTGTCGCCGTCGCCATCAGCGCTAACATCTCCGGCGGCCAC-

GTCAATCCCGCTGTCACGTTCGGCGCSTGCCTCGGCGGCCACATCACCATCCTACGTGGCC-

TACTCTACTGGATTGCCCAGTTGCTTGGCTCCGTCGSTCGCGTGCTTACTCCTCAAGSTTT-

GTCACCAATGGCATGACTACAACCGCTTTCGGTTTATCATCAGGAGTAAATGTATGGAAC-

GSTTTCGTAATGGAGATCGTATTGACCTTTGGGCTGGTCTATACCGTATACGCTACCGCACTG-

GATGGTAGGAAGGGCGAGTTGGGAATTATAGCACCAC-

TCGCGATCGGTCTCATAGTGGGGGCCAATATTTTGGTGGGSTGGGGSCCTTTGACGGAG-

CATCCATGAACCCGGCTGSTTTCSTTCGSCCCGGCCGTCSTGAGTTGGACTTGGGATAACCAC-

TGGATCTATTGGGCCGGGCCTCTTATTGGTAGTGCATTGGCTGCGATTATCTATGAGTT-

GTTCTTCATGAACCATACCCACGAGCAATTGCCCACCGCTGATTACTAA

The protein sequence encoded by the CsAQgP95 gene was specifi- cally shown in SEQ ID NO: 2 in the sequence listing:

MPMIYVDRITRRIAVGNREEATHPAALKAALAEFISTLIEFVFAGOGSGMAFNKITHSSET-

TPSGLIAAAITAHAFGLEFVAVAISANISGGHVNPAVTFGACLGGHITILRGLLYWIA-

QLLGSVVACLLLKEVTNGMTTTAFGLSSGVNVWNGEVMEIVLTFEGLVYTVYATALDGRKGEL-

GIILAPLAIGLIVGANILVGGAFDGASMNPAVSFGPAVVSWTWDNHWIYWAG-

PLIGSALAAIIYELFFMNHTHEQLPTADY

2. Expression analysis of the CsAQP25 gene {1) Expression patterns of the CsAQP95 gene in different tis- sues of tea plant

The tea plant cultivar C. sinensis 'Shuchazao' was planted in

Nongcuiyuan, Anhui Agricultural University, Luyang District, He- fei, Anhui Province. The 17 tissues and organs included bud, 1°% leaf, 1°" main vein, 2* leaf, 2™ main vein, 3% leaf, 3° main vein, 4* leaf, 4 main vein, 5% leaf, 5" main vein, vascular bundle, shoot between 1°" and 2" leaves (1-2 stem), stem between 2™ and 3% leaves (2-3 stem), stem between 3% and 4% leaves {3-4 stem), stem between 4™ and 5" leaves (4-5 stem), and roots. Also, these sam- ples were used for total RNA extraction and first-strand cDNA syn- thesis. The reverse transcription product (first-strand cDNA) was diluted 30-fold as a template, and a 20 pL reaction system was prepared using Hieff™ QPCR SYBR® Green Master Mix (No Rox) (Yeasen, Shanghai, China): 2.0 pL of 30-fold diluted reverse tran- scription product, 0.4 uL each of upstream and downstream primers (10 pmol/uL), 10 uL of Hieff™ qPCR SYBR® Green Master Miz, and 7.2

HL of ddH,0. Three replicates were prepared for each reaction. Sub- sequently, the following program was run on the Bio-rad CFX-96

Touch System: 1) initial denaturation at 95°C for 5 min; ii) 39 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s; and iii) from 65°C to 95°C, plotting the melting curve at 0.1°C/s. The upstream primer was 5'-

TGGCGGAGTTCATCTCAACC-3', and the downstream primer was 5'-

AGTAGGCCACGTAGGATGGT-3'. With tea plant CsGADPH gene as internal reference, based on the upstream primer (5'-TTGGCATCGTTGAGGGTCT- 3') and the downstream primer (5'-CAGTGGGRACACGGRAAGC-3'), the relative expression levels of CsAQP95 in different tissues of tea plant were calculated through the analysis software of the instru- ment. (2) Expression of tea plant CsAQP95 gene under different ni-

trogen treatments

Two-year-old tea cuttage seedlings (C. sinensis 'Shuchazao') were selected from a plant breeding base of Anhui Dechang Nursery

Stock Co., Ltd., Shucheng County, Anhui Province, China. The cut- ting seedlings of the same size were hydroponically cultivated in a greenhouse of the State Key Laboratory of Tea Plant Biology and

Utilization, Anhui Agricultural University. The greenhouse was set to a temperature of 25°C under a 16 h light/8 h dark cycle, and the relative humidity was set to 70-75%. First, the tea cutting seedlings were grown in basal nutrient solution for 1.5 months to obtain enough well-developed new roots of tea plant. Different forms of nitrogen treatments: one week after the nitrogen (N) de- ficiency treatment of the basal nutrient solution, the tea cutting seedlings were grown in an N-deficient solution and solutions sup- plemented with 1.43 mM Urea-N, 1.43 mM Ca (NO;).-N, and 1.43 mM (NH) 2504-N for 10 and 20 days, respectively. Root tissue samples were collected, quick-frozen in liquid nitrogen immediately, and stored in an ultra-low temperature freezer at -80°C for analysis of the expression of CsAQP95 gene. RNA extraction and quantitative

PCR assay were the same as above.

FIGS.1 and 2 illustrate expression patterns of CsAQP95 in different tissues of tea plant and under different forms of nitro- gen treatments, respectively. The quantitative PCR results of 14 different tissues and organs of C. sinensis 'Shuchazao' showed that tender tissues and roots with the expression pattern of

CsAQP95 had high expressive abundance. The results of hydroponic cultivation with different forms of nitrogen showed that expres- sion of CsAQP95 was significantly up-regulated 20 days after urea treatment. It is speculated that CsAQP95 may be involved in the absorption and transport of urea by tea plant roots. 3. Subcellular localization of tea plant CsAQP95

A pCAMBIA1305.1-CsAQPY95 vector was electroporated into EHA105

Agrobacterium Competent cells, and positive clones were identified by conventional PCR assay. A single clone correctly verified by colony PCR was picked, inoculated in 5 mL of LB broth (supplement- ed with 50 pg/mL rif and 100 ug/mL Spec), and cultured to OD = 0.8-1.2. 1 mL of Agrobacterium cells cultured overnight were inoc-

ulated into 100 mL of LB broth (supplemented with 50 pg/mL rif and 100 pg/mL Spec), and cultured at 200 r/min overnight at 28°C. The cells were collected by centrifugation, and resuspended to ODgyp = 0.4 with a resuspension that was adjusted to pH 5.6 using 10 mM

MgCl; and 10 mM 2-(N-morpholinc)ethanesulfonic acid. The bacterial suspension was incubated with 100 pM acetosyringone (As) for 2 h at 28°C, and mixed with EHA105 Agrobacterium ElectroCompetent cells at a ratio of 1:1 before injection. The bacterial suspension was aspirated by a disposable 1 mL syringe without needle, inject- ed from the lower epidermis of a tobacce leaf, and penetrated into the entire leaf tissue. After injection, the tobacco was treated in the dark for 8-12 h, and after culturing in a normal greenhouse for 2-3 days, the fluorescence of green fluorescent protein (GFP) was observed, recorded and photographed under a laser scanning confocal microscope.

FIG. 3 illustrates the subcellular localization of CsAQPS85 in tobacco epidemic leaf cells. As shown in FIG. 3, GFP represents green fluorescent protein; AtPIP2A::mCherry represents a plasma membrane maker gene; Bright Field represents a bright field image of pCAMBIA1305.1-CsAQP95; and Merged represents a merged image of

PCAMBIA1305.1-CsAQ9P95. As can be seen from FIG. 3: the two fluo- rescence signals of pCAMBIA1305.1-CsAQP95 (green fluorescence sig- nal of GFP) and AtPIP2A::mCherry (plasma membrane maker with red fluorescence signal) completely overlap, whereas the empty vector has signals in both nuclear and plasma membrane, and the signal and the nuclear localization signal does not overlap with the plasma membrane maker, indicating that the teaplant CsAQP95 is lo- calized at in the plasma membrane. 4. Functional verification of CsAQP95 gene in Arabidopsis thaliana (1) Transformation of pTCK303-CsAQ0P95 plasmid into Agrobacte- rium

The pTCK303-CsAQP95 plasmid that was sequenced correctly be- fore was taken, and 1 pL of the plasmid was electroporated into

EHA105 Agrobacterium Competent cells and sent to GE for sequencing and verification. (2) Genetic transformation into A. thaliana

A quantity of wild-type A. thaliana seeds were added with de- ionized water, vernalized in a 4°C refrigerator, and sown 72 h af- ter vernalization. After sowing, a preservative film was covered and placed under suitable conditions (humidity 60%; temperature 23°C; 16 h light/8 h dark cycle) to wait for germination. After the seeds germinated, seedlings of the same size were selected for transplanting and normal cultivation. The pTCK303-CsAQP95 vector was transformed into EHA105 Agrobacterium Competent cells by the freeze-thaw method, and positive clones were identified by conven- tional PCR assay. A positive colony containing a target gene was picked, and cultured in 5 mL of LB broth supplemented with corre- sponding antibiotics at 200 r/min for 24 h at 28°C; 2 mL of the cultured bacterial suspension was pipetted into 100 mL of fresh LB broth supplemented with corresponding antibiotics, and was sub- jected to further shake culture until ODgyy was approximately 1.0; cells were centrifugally collected, resuspended in a bwt% sucrose solution until the ODgy was approximately 0.8, and shaken well with a 0.1wt® silwet L-77. A. thaliana was planted for approxi- mately a month, and the plants began to bloom one after another; robust plants were selected as the plants to be transformed. Be- fore transformation, terminal inflorescences were continuously re- moved to make the plants produce more flower buds. The plants to be transformed needed to be fully watered one day before transfor- mation.

The prepared transformation solution was filled in a contain- er. The A. thaliana inflorescence was soaked in the transformation solution for approximately 30 s, left to stand in the dark for 24 h, and cultivated normally to harvest the seeds. The harvested A. thaliana seeds were placed in a centrifuge tube, sterilized with 1 mL of 75% ethanol for 1 min and 10% NaClO for 5 min, and rinsed with sterile water 5-6 times. The seeds were aspirated with a pi- pette tip and sown on MS agar. The seeds were vernalized in the dark for 72 h at 4°C, and transferred to a culture chamber at 23°C; the seeds were cultured under a 16 h light/8 h dark cycle.

Approximately two weeks later, the resistant plants with green leaves and normal root development were selected and transplanted into the cultivation substrate for further cultivation. Before transplantation, the cultivation substrate absorbed water suffi- ciently. A preservative film was covered after transplantation and removed after approximately 3 days. Afterwards, the management was the same as above, and T2 seeds were harvested for experiments.

The DNA and RNA of A. thaliana were extracted at the seedling stage, and the expression of the target gene was detected by PCR using gene-specific primers. The transgenic plants were cultured for 2 h at -6°C, taken out of the Petri dish, cultured in the dark for 12 h at 4°C, and transferred to a normal culture chamber for culture, and the survival of the seedlings was observed after 4 days.

FIG. 4 illustrates growth phenotypes of wild-type and CsAQP95 transgenic A. thaliana. As shown in FIG. 4, the plant height, bio- mass and seed weight were significantly improved in the CsAQP95- overexpressed line (CsAQP95-0OE) plants and the complemented A. thaliana mutant line (Ubi:CsAQP95/atdur3) compared with the wild type, indicating that the expression of CsAQP35 could significant- ly increase the biomass and yield of A. thaliana plants, providing a theoretical basis for improving the nitrogen uptake and utiliza- tion efficiency of tea plants and increasing shoot weight by mo- lecular-assisted breeding, as well as functional gene resources.

The present disclosure only provides an example of A. thali- ana, and other plants are also suitable for the method provided by the present disclosure.

The above content is only an example and description of the structure of the present disclosure. Those skilled in the art can make various modifications or supplements to the specific example described or replace them in a similar manner, as long as they do not depart from the structure of the present disclosure or go be- yond the scope defined by the claims, all of which fall within the protection scope of the present disclosure.

Sequence Listing <110> Anhui Agricultural University <120> TEA PLANT AQUAPORIN GENE CsAQP95 AND APPLICATION THEREOF <130> NO <160> 2 <170> SIPOSegquenceListing 1.0 <210> 1 <211> 777 <212> DNA <213> Tea plant (Camellia sinensis L. O. Kuntze) <400> 1 atgccgatga tctacgtgga tcggattact cgccggatcg cggtcggaaa ccgggaagag 60 gegacccacc ccgcegctct caaggcggcg ctggcggagt tcatctcaac cctaatttte 120 gtettegegg gccagggatc cgggatggcc ttcaataaga tcactcatag cagcttcact 180 accccctceg gcctcatcgc cgccgctatt gcccacgcat tcggactttt tgtegccgte 240 gccatcagcg ctaacatctc cggcggccac gtcaatcceg ctgtcacgtt cggcgcgtgec 300 cteggcggcc acatcaccat cctacgtgge ctactctact ggattgccca gttgettgge 360 teegtegteg cgtgcttact cctcaagttt gtcaccaatg gcatgactac aaccgctttc 420 ggtttatcat caggagtaaa tgtatggaac ggtttcgtaa tggagatcgt attgaccttt 480 gggctggtect ataccgtata cgctaccgca ctggatggta ggaagggcga gttgggaatt 540 atagcaccac tcgcgatcgg tctcatagtg ggggccaata ttttggtggg tggggccttt 600 gacggagcat ccatgaaccc ggctgttteg ttcggcccgg ccgtcgtgag ttggacttgg 660 gataaccact ggatctattg ggcegggcet cttattggta gtgcattggc tgcgattatec 720 tatgagttgt tcttcatgaa ccatacccac gagcaattgc ccaccgctga ttactaa 177 <210> 2 <211> 258 <212> PRT <213> Tea plant (Camellia sinensis L. O. Kuntze) <400> 2

Met Pro Met Ile Tyr Val Asp Arg Ile Thr Arg Arg Ile Ala Val Gly 1 5 10 15

Asn Arg Glu Glu Ala Thr His Pro Ala Ala Leu Lys Ala Ala Leu Ala 20 25 30

Glu Phe Ile Ser Thr Leu Ile Phe Val Phe Ala Gly Gln Gly Ser Gly 35 40 45

Met Ala Phe Asn Lys Ile Thr His Ser Ser Phe Thr Thr Pro Ser Gly 60

Leu Ile Ala Ala Ala Ile Ala His Ala Phe Gly Leu Phe Val Ala Val 65 70 75 80

Ala Ile Ser Ala Asn Ile Ser Gly Gly His Val Asn Pro Ala Val Thr 85 30 95

Phe Gly Ala Cys Leu Gly Gly His Ile Thr Ile Leu Arg Gly Leu Leu 100 105 110

Tyr Trp Ile Ala Gln Leu Leu Gly Ser Val Val Ala Cys Leu Leu Leu 115 120 125

Lys Phe Val Thr Asn Gly Met Thr Thr Thr Ala Phe Gly Leu Ser Ser 130 135 140

Gly Val Asn Val Trp Asn Gly Phe Val Met Glu Ile Val Leu Thr Phe 145 150 155 160

Gly Leu Val Tyr Thr Val Tyr Ala Thr Ala Leu Asp Gly Arg Lys Gly 165 170 175

Glu Leu Gly Ile Ile Ala Pro Leu Ala Ile Gly Leu Ile Val Gly Ala 180 185 190

Asn Ile Leu Val Gly Gly Ala Phe Asp Gly Ala Ser Met Asn Pro Ala 195 200 205

Val Ser Phe Gly Pro Ala Val Val Ser Trp Thr Trp Asp Asn His Trp 210 215 220

Ile Tyr Trp Ala Gly Pro Leu Ile Gly Ser Ala Leu Ala Ala Ile Ile 225 230 235 240

Tyr Glu Leu Phe Phe Met Asn His Thr His Glu Gln Leu Pro Thr Ala 245 250 255

Asp Tyr

JYXP20221100889 CN 202210475300.X 2022-04-29 Anhui Agricultural University

TEA PLANT AQUAPORIN GENE CsAQP95 AND APPLICATION THEREOF 8 777 DNA

PAT source 1..777 mol_type other DNA organism Tea tree (Camellia sinensis L. O.

Kuntze) atgccgatgatctacgtggatcggattactcgccggatcgcggtcggaaaccgggaagaggcgacccaccccgccg ctctcaaggcggcgctggcggagttcatctcaaccctaattttcgtcttcgcgggccagggatccgggatggccttcaat aagatcactcatagcagcttcactacccccteccggcctcatcgccgccgctattgcccacgcattcggactttttgtcgcc gtcgccatcagcgctaacatctccggcggccacgtcaatcccgctgtcacgttcggcgcgtgcctcggcggccacatca ccatcctacgtggcctactctactggattgcccagttgcttggctcegtcgtcgcgtgcttactcctcaagtttgtcaccaa tggcatgactacaaccgctttcggtttatcatcaggagtaaatgtatggaacggtttcgtaatggagatcgtattgacct ttgggctggtctataccgtatacgctaccgcactggatggtaggaagggcgagttgggaattatagcaccactcgcga tcggtctcatagtgggggccaatattttggtgggtggggcctttgacggagcatccatgaacccggctgtttcgttcggc ccggccgtcgtgagttggacttgggataaccactggatctattgggccgggcctcttattggtagtgcattggctgcgat tatctatgagttgttcttcatgaaccatacccacgagcaattgcccaccgctgattactaa 258 AA PAT source 1..258 mol_type protein organism Tea tree (Camellia sinensis L. O. Kuntze)

MPMIYVDRITRRIAVGNREEATHPAALKAALAEFISTLIFVFAGQGSGMAFNKITHSSFTTPSGL

IAAAIAHAFGLFVAVAISANISGGHVNPAVTFGACLGGHITILRGLLYWIAQLLGSVVACLLLKF

VTNGMTTTAFGLSSGVNVWNGFVMEIVLTFGLVYTVYATALDGRKGELGIIAPLAIGLIVGANI

LVGGAFDGASMNPAVSFGPAVVSWTWDNHWIYWAGPLIGSALAAIIYELFFMNHTHEQLPTA

DY 27 DNA PAT misc_feature 1..27 note upstream primer for PCR amplication of

CsAQP95 gene source 1..27 mol_type other DNA organism synthetic construct ggggtaccatgccgatgatctacgtgg 27 DNA PAT misc_feature 1..27 note downstream primer for PCR amplication of CsAQP95 gene source 1..27 mol_type other DNA organism synthetic construct ggactagtttagtaatcagcggtgggc 20 DNA PAT misc_feature 1..20 note upstream primer for PCR source 1..20 mol_type other DNA organism synthetic construct tggcggagttcatctcaacc 20 DNA PAT misc_feature 1..20 note downstream primer for PCR source 1..20 mol_type other DNA organism synthetic construct agtaggccacgtaggatggt 19 DNA PAT misc_feature 1..19 note upstream primer for PCR source 1..19 mol_type other DNA organism synthetic construct ttggcatcgttgagggtct 19 DNA PAT misc_feature 1..19 note downstream primer for PCR source 1..19 mol_type other DNA organism synthetic construct cagtgggaacacggaaagc

Claims

CONCLUSIESCONCLUSIONS

1. Aguaporine gen CsAQP95 van de theeplant, waarbij het aquaporine gen CsAQP95 van de theeplant een nucleotideseguentie heeft die wordt weergegeven in SEQ ID NO: 1 in de sequentielijst.1. Tea plant aguaporin gene CsAQP95, wherein the tea plant aquaporin gene CsAQP95 has a nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing.

2. Aquaporine gen CsAQgP95 van de theeplant volgens conclusie 1, waarbij een eiwitsequentie die wordt gecodeerd door het aquaporine gen CsAQP95 van de theeplant wordt weergegeven in SEQ ID NO: 2 in de sequentielijst.The tea plant aquaporin gene CsAQgP95 according to claim 1, wherein a protein sequence encoded by the tea plant aquaporin gene CsAQP95 is shown in SEQ ID NO: 2 in the sequence listing.

3. Expressievector pTCK303-CsAQ9P95 van de theeplant-, waarbij de expressievector wordt verkregen door enzymdigestie van een frag- ment dat wordt getoond in SEQ ID NR: 1 naar een pTCK303-vector.3. Tea plant expression vector pTCK303-CsAQ9P95, wherein the expression vector is obtained by enzyme digestion of a fragment shown in SEQ ID NO: 1 into a pTCK303 vector.

4. Toepassing van een aquaporine gen CsAQP95 van een theeplant bij het vergroten van de biomassa van planten.4. Application of an aquaporin gene CsAQP95 from a tea plant in increasing plant biomass.

5. Werkwijze voor het vergroten van de biomassa van planten met behulp van een aquaporine gen CsAQP95 van een theeplant, omvat- tende de volgende stappen: stap 1, het bereiden van een transformatieoplossing: het trans- formeren van een pTCK303-CsAQP95 vector in EHA105 Agrobacterium ElectroCompetent-cellen door middel van een vries-ontdooimethode, en het identificeren van een positieve kloon door middel van een conventionele PCR-assay om de transformatieoplossing te bereiden; en stap 2, bloeiwijze van een plant in de transformatie-oplossing we- ken, 24 uur in het donker laten staan voor normale kweek, zaden oogsten, geoogste zaden in een centrifugebuis plaatsen, sterilis- eren, de zaden op MS-agar trekken met behulp van een pipetpunt, verniseren van de zaden in het donker bij 4 °C gedurende 72 uur, de zaden overbrengen naar een kweekkamer bij 23 °C; het kweken van de zaden onder een cyclus van 16 uur licht/8 uur donker gedurende twee weken, het selecteren en verplanten van resistente planten met groene bladeren en normale wortelontwikkeling in een kweeksub-5. Method for increasing plant biomass using an aquaporin gene CsAQP95 from a tea plant, comprising the following steps: step 1, preparing a transformation solution: transforming a pTCK303-CsAQP95 vector into EHA105 Agrobacterium ElectroCompetent cells by a freeze-thaw method, and identifying a positive clone by a conventional PCR assay to prepare the transformation solution; and step 2, soak inflorescence of a plant in the transformation solution, leave in the dark for 24 hours for normal cultivation, harvest seeds, place harvested seeds in a centrifuge tube, sterilize, pull the seeds onto MS agar with using a pipette tip, varnish the seeds in the dark at 4°C for 72 hours, transfer the seeds to a growth chamber at 23°C; growing the seeds under a cycle of 16 hours light/8 hours dark for two weeks, selecting and transplanting resistant plants with green leaves and normal root development in a growing sub-

straat voor verdere kweek, het kweeksubstraat volledig water laten opnemen voor het verplanten, afdekken met een conserveringsfilm na verplanten en verwijderen van de conserveringsfilm voor kweek na drie dagen, waarbij de biomassa van verkregen T2-zaden wordt ver- beterd.street for further cultivation, allow the cultivation substrate to completely absorb water before transplanting, cover with a preservation film after transplanting and remove the preservation film for cultivation after three days, thereby improving the biomass of T2 seeds obtained.

6. Werkwijze voor het vergroten van de biomassa van planten met behulp van een aquaporine gen CsAQP25 van een theeplant volgens conclusie 5, waarbij in stap 1 de transformatieoplossing specifiek wordt bereid volgens de volgende stappen: het plukken van een pos- itieve kolonie die een doelgen omvat, en het kweken van het posi- tieve kolonie in een LB-bouillon aangevuld met overeenkomstige an- tibiotica bij 28 °C gedurende 24 uur; pipetteren van een gekweekte bacteriële suspensie in een verse LB-bouillon aangevuld met overeenkomstige antibiotica, en het verder uitvoeren van schudk- week totdat ODsse 1,0 is; het centrifugaal verzamelen van cellen, het hersuspenderen van de cellen in een 5 gew.% sucrose-oplossing totdat de ODgss 0,8 is; en de cellen goed schudden met een 0,1 gew.% organosilicium surfactant om de transformatieoplossing te verkrijgen.A method for increasing plant biomass using an aquaporin gene CsAQP25 from a tea plant according to claim 5, wherein in step 1 the transformation solution is specifically prepared according to the following steps: picking a positive colony carrying a target gene and culturing the positive colony in an LB broth supplemented with corresponding antibiotics at 28°C for 24 hours; pipetting a cultured bacterial suspension into a fresh LB broth supplemented with corresponding antibiotics, and continuing shaking culture until ODsse is 1.0; collecting cells centrifugally, resuspending the cells in a 5 wt% sucrose solution until the ODgss is 0.8; and shake the cells well with a 0.1 wt% organosilicon surfactant to obtain the transformation solution.

7. Werkwijze voor het vergroten van de biomassa van planten met behulp van een aquaporine gen CsAQP25 van een theeplant volgens conclusie 5, waarbij in stap 2 een sterilisatiewerkwijze als volgt is: steriliseren van de zaden met 75% (v/v) ethanol gedurende 1 min en met 10wt % NaClO:; gedurende 5 min, en het 5-6 keer spoelen van de zaden met steriel water.A method for increasing the biomass of plants using an aquaporin gene CsAQP25 from a tea plant according to claim 5, wherein in step 2 a sterilization method is as follows: sterilizing the seeds with 75% (v/v) ethanol for 1 min and with 10wt% NaClO:; for 5 min, and rinsing the seeds 5-6 times with sterile water.