CN116024250A - Sweet potato leaf development related protein IbNAC43, and coding gene and application thereof - Google Patents

Abstract

The invention discloses an IbNAC43 gene derived from sweet potato, and a coded protein and application thereof. The IbNAC43 gene encodes a protein that is A1, A2 or A3 as follows: a1, the amino acid sequence is protein of the amino acid sequence shown as SEQ ID No.2 in the sequence table; a2, carrying out substitution and/or deletion and/or addition of amino acid residues on an amino acid sequence shown in SEQ ID No.2 in a sequence table to obtain a protein which has more than 80% of identity with the protein shown in A1) and is related to plant leaf development; a3, N-terminal or/and C-terminal of A1) or A2). According to the invention, the coding gene of the IbNAC43 protein is introduced into the sweet potato, so that the leaf curl index of the plant is increased, the chlorophyll content is reduced, the photosynthesis rate is reduced, and the number of epidermal cells under the leaf is obviously increased compared with that of the upper epidermis.

Description

Sweet potato leaf development related protein IbNAC43, and coding gene and application thereof

Technical Field

The invention relates to a sweet potato leaf development related protein IbNAC43 in the biotechnology field and a coding gene and application thereof.

Background

The leaf is one of the important components of plant organ, is the main place for photosynthesis, respiration and transpiration of plant, and is used in synthesizing plant matter and controlling the exchange of water in plant body with outer environment and can affect the growth and yield of crop. Practical studies have found that severe leaf morphology changes can inhibit the growth of plants, and that proper crimping of leaves can reduce water loss and enhance stress resistance of plants. Sweet potato (Ipomoea batatas (l.) lam.) is an important crop for food, feed and industrial raw materials, is a novel energy plant, and is one of the most economical and effective ways for improving the yield of sweet potato by researching the plant leaf development mechanism.

Sweet potato is a vegetative propagation crop, and the resource utilization and parent free assembly in sweet potato breeding are severely limited by interspecific and intraspecific hybridization incompatibility. Long-term breeding practices show that it is difficult to select a new sweet potato variety with high quality, high yield and drought resistance by using a conventional hybridization breeding method. Whereas NAC genes are an important family of transcription factors in plants, involved in plant growth and development and in the stress response process. At present, research on the influence of NAC protein in sweet potato on leaf development has not been reported.

Disclosure of Invention

The invention aims to solve the technical problem of how to promote the development of sweet potato leaves so as to improve the yield.

Any of the following applications of IbNAC43 protein or a substance that modulates the content or activity of said IbNAC43 protein:

d1 Regulating and controlling plant leaf curl index;

d2 Application of regulating chlorophyll content in plant leaves;

d3 Regulating and controlling the photosynthetic rate of the plant leaves;

d4 Regulating plant leaf morphology;

d5 Regulating the relative ratio of the number of the upper and lower epidermal cells of the plant leaf;

d6 Regulating plant leaf development;

d7 Performing plant breeding applications;

the IbNAC43 protein is the protein of A1, A2 or A3 as follows:

a1, the amino acid sequence is protein of the amino acid sequence shown as SEQ ID No.2 in the sequence table;

a2, carrying out substitution and/or deletion and/or addition of amino acid residues on an amino acid sequence shown in SEQ ID No.2 in a sequence table to obtain a protein which has more than 80% of identity with the protein shown in A1) and is related to plant leaf development;

a3, N-terminal or/and C-terminal of A1) or A2).

In the above application, SEQ ID No.2 of the sequence Listing consists of 372 amino acid residues.

In the above applications, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In the above applications, the 80% identity or more may be at least 81%, 85%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above application, the IbNAC43 protein may be derived from sweet potato.

In the above application, the plant is a monocotyledonous plant or a dicotyledonous plant; further, the monocot plant may be a Convolvulaceae plant; still further, the Convolvulaceae plant may be a sweet potato plant; specifically, the sweet potato plant may be sweet potato.

In the above application, the substance that modulates the content or activity of IbNAC43 protein may be a substance that performs at least one of the following 6 modulations: b1 Regulation at the level of transcription of said gene; b2 Regulation after transcription of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); b3 Regulation of RNA transport of the gene (i.e., regulation of nuclear to cytoplasmic transport of mRNA of the gene); b4 Regulation of translation of said gene; b5 Regulation of mRNA degradation of said gene; b6 Post-translational regulation of the gene (i.e., regulation of the activity of the protein translated by the gene).

In the above application, the substance regulating the content or activity of the IbNAC43 protein is a biological material related to the IbNAC43 protein; the biological material is any one of the following C1-C3:

c1, a nucleic acid molecule encoding the IbNAC43 protein;

c2, a nucleic acid molecule that increases expression of the IbNAC43 protein;

c3, expression cassette comprising a nucleic acid molecule as described in C1 or C2, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ.

In the above application, the nucleic acid molecule of C1 or C2 may be DNA such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above application, the nucleic acid molecule C1 may specifically be a nucleic acid molecule whose coding sequence of the coding strand is positions 1-1119 of SEQ ID No.1 in the sequence Listing.

In this application, the modulation may be up-regulation or enhancement or increase, or down-regulation or inhibition or decrease.

In the above application, the plant is any one of the following:

c1 Dicotyledonous or monocotyledonous plants;

c2 A plant of the order of the tubular flower,

c3 A plant of the family Convolvulaceae,

c4 A plant of the genus sweet potato,

c5 Sweet potato.

The present invention also provides a protein, ibNAC43 protein, which is A1, A2 or A3 protein as follows:

a1, the amino acid sequence is protein of the amino acid sequence shown as SEQ ID No.2 in the sequence table;

a2, carrying out substitution and/or deletion and/or addition of amino acid residues on an amino acid sequence shown in SEQ ID No.2 in a sequence table to obtain a protein which has more than 80% of identity with the protein shown in A1) and is related to plant leaf development;

a3, N-terminal or/and C-terminal of A1) or A2).

The invention also provides biological materials related to the IbNAC43 protein; the biological material is any one of the following C1-C3:

c1, a nucleic acid molecule encoding the IbNAC43 protein;

c2, a nucleic acid molecule that increases expression of the IbNAC43 protein;

c3, expression cassette comprising a nucleic acid molecule as described in C1 or C2, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ.

Herein, the object of plant breeding may include growing plants with altered leaf polarity development.

The change in blade polarity development may be embodied as

E1 Plant leaf curl index increases;

d2 Application of regulating chlorophyll content in plant leaves;

d3 Regulating and controlling the photosynthetic rate of the plant leaves;

d4 Regulating plant leaf morphology;

d5 Regulating the relative ratio of the number of the upper and lower epidermal cells of the plant leaf;

herein, the plant may be any one of the following plants:

e1 A plant of the class monocotyledonae,

e2 A plant of the family Convolvulaceae,

e3 A plant of the genus sweet potato,

e4 Sweet potato.

In the present invention, the regulation may be up-regulation or enhancement or improvement, or down-regulation or inhibition or reduction.

The invention discovers an IbNAC43 protein and a coding gene thereof, introduces the gene into a sweet potato cultivar to obtain a sweet potato plant transformed with the IbNAC43 gene, carries out drought stress treatment on the transgenic sweet potato plant, and discovers that compared with a wild cultivar, the leaf curl is obvious, and the leaf curl is particularly characterized by higher leaf curl index, plant dwarfing, chlorophyll content reduction, leaf photosynthetic rate reduction and lignin content increase. The results show that the IbNAC43 gene and the protein encoded by the gene play an important role in the normal growth and development of plants. The IbNAC43 protein and the coding gene thereof provided by the invention have important application value in the growth and development process of plant leaves. The invention has a certain application space and market prospect in the agricultural field.

Drawings

FIG. 1 shows the result of PCR amplification of a part of transgenic sweetpotato plants in example 2 of the present invention. Wherein M is DNA molecule Marker, W is negative control water, P is positive control pCAMBIA1300-IbNAC43 vector plasmid DNA, WT is genomic DNA of wild potato 19 plant, L1, L2, L3, L5 are genomic DNA of IbNAC43 gene-transferred negative plant; l4, L6 and L7 are genomic DNA of IbNAC43 gene-positive plants.

FIG. 2 shows the expression level of the IbNAC43 gene in the overexpressing transgenic sweetpotato plants and wild-type plants (WT) according to example 2 of the present invention; wherein, WT is cDNA of wild potato 19 plant, L4, L6, L7, L10, L12 and L14 are cDNA of IbNAC43 gene-transferred sweet potato positive plant. The figure shows that P <0.01 is the result of the significance analysis of the differences from the wild type.

FIG. 3 shows the growth of a transgenic IbNAC43 sweet potato plant and a wild type plant according to example 2 of the invention under normal growth conditions; wherein, the A diagram of FIG. 3 is a plant phenotype photograph (the whole plant growth state of sweet potato plants is sequentially shown from top to bottom, the front surface of the leaf and the cross section thereof); FIG. 3B is a graph showing blade curl index statistics; FIG. 3C is the relative chlorophyll content measurement result; FIG. 3D is a graph showing the results of photosynthetic rate measurement; WT is a wild-type sweetpotato plant, L4, L10 and L14 are over-expressed IbNAC43 transgenic sweetpotato lines. The figure shows that P <0.01 is the result of the significance analysis of the differences from the wild type.

FIG. 4 is a leaf histology observation of a transgenic IbNAC43 sweet potato plant and a wild type plant according to example 2 of the invention; wherein, the A diagram of FIG. 4 is a scanning electron microscope observation diagram of the front side (ad) and the back side (ab) of the blade; FIG. 4B is a view of the blade from a transverse direction; panel C of FIG. 4 shows the relative ratio of leaf upper and lower epidermal cell numbers; wherein, WT is wild sweet potato plant, L10, L14 are the transgenic sweet potato line of overexpression IbNAC43. The figure represents the results of the significance analysis of differences from the wild type P <0.05.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The quantitative tests in the following examples were all performed in triplicate, and the results were averaged.

The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The sweet potato closely related wild variety Ipomoea trifida in the examples below is described in the following literature: wei Song identification and analysis of drought-resistance-related NAC transcription factor of Ipomoea trifida (2 x) as a closely related wild species of Ipomoea batatas, university of agricultural university of China, university of Shuo's treatises, 2017. The public is available from the national agricultural university to repeat the experiment.

Sweet potato 19 in the following examples is described in the following documents: ren Zhitong characterization and molecular mechanism analysis of sweetpotato plants overexpressing the IbbEFP and IbSnRK1 genes. The public is available from the national agricultural university to repeat the experiment.

The cloning vector pMD19-T in the following examples is a product of Takara Bio-engineering (Dalian) under the product catalog number 6013.

Vector pCAMBIA1300 in the examples described below is a product of Cambia corporation.

The total plant RNA extraction kit in the following examples is a Transzol total plant RNA extraction kit (catalog number: ET 101) of the full gold company (TransGen Biotech, beijing).

PrimeScript in the following examples ^TM RT reagent Kit with gDNA Eraser the kit is a product (catalog number: RR 047A) from TaKaRa (Takara Bio (engineering) Inc., dalian).

The following examples use SPSS statistical software to process the data, and the experimental results are expressed as mean.+ -. Standard deviation, using Student t-test, with P <0.05 (x) indicating significant differences and P <0.01 (x) indicating very significant differences.

Example 1 acquisition of sweetpotato leaf development-related protein IbNAC43 and Gene encoding the same

1. Protein IbNAC43 related to sweet potato leaf development and obtaining of coding gene thereof

Experimental materials: the young leaves of the developed leaves are taken down, quick frozen by liquid nitrogen and preserved at-80 ℃.

1. Obtaining cDNA templates

Extracting total RNA of tender leaves of Ipomoea trifida, a closely related wild variety of Ipomoea, with a plant total RNA extraction kit, and subjecting the total RNA to PrimeScript ^TM RT reagent Kit with gDNA Eraser the kit reverse transcribes the first strand cDNA.

2. And (3) taking the cDNA obtained in the step (1) as a template, designing specific primers 1 and 2 according to a homologous cloning method, and carrying out PCR amplification, wherein the sequences of the primers are as follows:

primer 1:5'-ATGAATCTTTCCGTGAATGGTC-3';

primer 2:5'-CTATACCGACAAGTGGGACAAGG-3'.

The amplified fragment obtained was ligated to the cloning vector pMD19-T to give recombinant plasmid 2. Sequencing the recombinant plasmid 2 to obtain the nucleotide sequence of the amplified fragment.

3. The nucleotide sequence of the IbNAC43 gene is obtained according to the sequencing result of the step 2. The gene shown in the sequence is named IbNAC43, and the coding sequence (ORF) of the gene is SEQ ID NO.1, nucleotide 1-1119 from the 5' end; the protein coded by the gene is named IbNAC43, the amino acid sequence of the protein is shown as SEQ ID NO.2, and the protein consists of 372 amino acid residues.

SEQ ID NO.1 is as follows:

atgaatcttt ccgtgaatgg tcagtcccgg gttcctcccg gattccggtt ccaccccacc 60

gaggaagagc tcctccacta ctacctccgg aagaaggtca cctccgagaa gattgacctc 120

gacgtcattc gcgacgtcga tctcaacaag cttgagtcct gggatattca agagaagtgc 180

aagataggat cggggccgca gaacgactgg tactttttca gccacaagga caagaagtat 240

ccgacaggaa cccggacaaa ccgggcgacg gcggccggat tctggaaggc caccgggagg 300

gataaggtga tatacagcaa ctgcaagaga atcggtatga gaaaaacctt agtattctac 360

aaaggtcgag cccctcacgg acaaaaatct gattggatca tgcatgagta caggttggac 420

gacaattccc ctgaacccac cggcacaagt ttggtcggag aattgaacag tgcaccggaa 480

gaaggttggg tggtctgccg tgtcttcaag aagaaaaact atcacaaggc tcttgagagt 540

ccccataccg ttgttcaaac ccatcctgga aacgacggcg ttctggatca aatccttact 600

tacatgggaa ggtcatcttc gtcatccaag caacaaaacg gatccaaaaa cggcaatctt 660

ataatcaacg gcgacgatac ccccatgcaa ttctcccatg caatccaggc cgcccgattc 720

ctccaccttc cgcccctgga aacccaatcc gccatggaaa tcctctcttc tcccacccac 780

gcctccttcg aagacatgct ggccgaaccc gaaccgtcct gcaacaacac tgaaccggtc 840

gacgatatga aaaccggacc cggtccagcc gactgggttg ctctggaccg cctcgtcgcc 900

tctcagctca acggccagat cgaatcctcc aaaagctacg tggccgatga cctcagcgac 960

ggcctgtatt ttcccatcgt cgatcaccac cgtccgtttg gcagaccaaa taatcatcac 1020

cacaactctc aggtcaacgc cggcaacgag gttgaattct ggagctacac caactcctca 1080

tcatcaccgt ccgatccctt gtcccacttg tcggtatag 1119

SEQ ID NO.2 is as follows:

MNLSVNGQSQVPPGFRFHPTEEELLHYYLRKKVTSEKIDLDVIRDVDLNKLEPWDIQEKCKIGSGPQNDWYFFSHKDKKYPTGTRTNRATAAGFWKATGRDKVIYSNCKRIGMRKTLVFYKGRAPHGQKSDWIMHEYRLDDNSPEPTGTSLVGELNSAPEEGWVVCRVFKKKNYHKALESPHTVVQTHPGNDGVLDQILTYMGRSSSSSKQQNGSKNGNLIINGDDTPMQFSHAIQAARFLHLPPLETQSAMEILSSPTHASFEDMLAEPEPSCNNTEPVDDMKTGPGPADWVALDRLVASQLNGQIESSKSYVADDLSDGLYFPIVDHHRPFGRPNNHHHNSQVNAGNEVEFWSYTNSSSSPSDPLSHLSV

example 2 use of IbNAC43 protein in influencing plant leaf development

1. Construction of recombinant plasmid pCAMBIA1300-IbNAC43

1. The double-stranded DNA molecule shown in positions 1-1116 from the 5' -end of SEQ ID NO.1 is artificially synthesized. The double-stranded DNA molecule is used as a template, and the primer 3 and the primer 4 are used for PCR amplification, wherein the primer sequences are as follows:

primer 3:

5’-ACGGGGGACGAGCTCGGTACCATGAATCTTTCCGTGAATGGT-3' (underlined indicates the recognition sequence for restriction endonuclease Kpn I);

primer 4:5' -GCTCACCATGTCGACTCTAGATACCGACAAGTGGGACAAGG-3' (underlined indicates the recognition sequence for the restriction endonuclease Xba I).

The amplified product was recovered to obtain a double-stranded DNA molecule comprising the recognition sequence of restriction enzyme Kpn I at one end and the recognition sequence of restriction enzyme Xba I at the other end, which was designated as fragment 1.

2. After completion of step 1, the vector pCAMBIA1300-GFP was digested with the restriction enzymes Kpn I and Xbal I, and the vector was described in the following literature: luo H, meng D, liu H, xie M, yin C, liu F, dong Z, jin W.Ectopic expression of the transcriptional regulator silky3 causes pleiotropic meristem and sex determination defects in maize inflo relices.plant cell.2020Dec;32 (12) 3750-3773. A vector backbone of about 10449bp, designated vector backbone 2, was recovered.

3. The fragment 1 was ligated to the vector backbone 2 using homologous recombination cloning enzyme (Hieff Clone Universal One Step Cloning Kit, YEASEN Co.) at 50℃for 20min to give a recombinant plasmid pCAMBIA1300-IbNAC43, which was obtained by replacing the fragment (small fragment) between the recognition sites of restriction endonucleases KpnI and Xba I of pCAMBIA1300-GFP vector with a double-stranded DNA molecule whose coding strand nucleotides were shown at positions 1-1116 of SEQ ID NO.1, keeping the other sequences of pCAMBIA1300-GFP vector unchanged.

Recombinant plasmid pCAMBIA1300-IbNAC43 expresses IbNAC43 protein shown in SEQ ID NO.2 of the sequence Listing.

The recombinant plasmid pCAMBIA1300-IbNAC43 is transformed into escherichia coli DH5a (purchased from Beijing full gold biotechnology Co., ltd., product catalog number is CD 201-01), and the recombinant vector is cultured for 20 hours at 37 ℃, subjected to PCR analysis and enzyme digestion identification, and subjected to sequencing verification. Sequencing results show that the sequence shown in 1 st to 1116 st positions of the 5' end of SEQ ID NO.2 is inserted between Kpn I and Xbal I cleavage sites of the vector pCAMBIA1300, which proves that the recombinant vector is constructed correctly.

2. Obtaining transgenic plants of sweet potato

1. The recombinant plasmid pCAMBIA1300-IbNAC43 is transformed into agrobacterium tumefaciens EHA105 to obtain recombinant agrobacterium, and the recombinant agrobacterium is named EHA105/pCAMBIA1300-IbNAC43.

2. Culturing agrobacterium: after the agrobacterium EHA105/pCAMBIA1300-IbNAC43 bacterial liquid is activated, single colonies are picked and inoculated into 50mL of LB liquid medium added with kanamycin (final concentration of 50 mg/L) and rifampicin (final concentration of 25 mg/L), and the culture medium is subjected to shaking culture at 200rpm at 28 ℃ until the OD600 value is 0.6-0.8. Centrifugation at 5000rpm and removal of supernatant, resuspension of the cells with an appropriate volume of liquid MS medium containing 2,4-D (2.0 mg/L) at an OD600nm of about 0.8 yielded EHA105/pCAMBIA1300-IbNAC43 Agrobacterium suspension.

3. Infection:

harvested potato pieces of the potato 19 (wild type) are used for providing sweet potato stem tips, stripped stem tip meristems are inoculated on a callus induction solid medium (MS solid medium containing 2.0 mg/L2, 4-D), dark culture is carried out for 8 weeks at room temperature of 27+/-1 ℃, callus induction is carried out, and then embryogenic callus induction liquid medium (MS liquid medium containing 2.0 mg/L2, 4-D) is used for propagation and subculture, so that a potato 19 embryogenic cell suspension system is established for transformation.

And (3) selecting and grinding the potato 19 embryogenic cell suspension line subjected to the secondary culture for 6-8 weeks in an ultra clean bench for 3d, suspending embryogenic suspension cell masses with diameters of 0.7-1.4mm in the EHA105/pCAMBIA1300-IbNAC43 agrobacterium heavy suspension prepared in the step (2), sucking out liquid after 5min, and then co-culturing for 3d under the dark condition of 27+/-1 ℃.

4. Delay culture: transferring the suspension cells after 3D co-culture in the step 3 into an MS liquid culture medium containing 2 mg/L2, 4-D and 300mg/L cephalosporin (cefotaxime sodium, CS) for 3 times, washing out agrobacterium tumefaciens adsorbed on the surface of cell mass during infection as much as possible, and then continuing to culture in the MS liquid culture medium containing 2 mg/L2, 4-D and 300mg/L cephalosporin (cefotaxime sodium, CS) for 1 week.

5. Screening and culturing: the cell mass after the delay culture was gently transferred and plated on filter paper on MS solid medium containing 2 mg/L2, 4-D, 300mg/L Cephalosporin (CS) and 5mg/L Hygromycin (Hygromycin B, hyg), and the selection culture was performed in the dark at 27.+ -. 1 ℃. The selection medium was then changed 1 time every 2 weeks.

6. Regeneration of plants: after 8 weeks of selective culture, transferring the callus with good growth state in the screening culture medium to MS solid culture medium containing 1.0mg/L ABA and 300mg/L CS for induction of somatic embryo under the conditions of 27+ -1deg.C, 13h illumination every day and 3000Lx illumination intensity.

7. Obtaining a quasi-transgenic plant: mature somatic embryos which turn green on an ABA culture medium after induction of 2-4w are transferred to an MS solid culture medium and are cultured for 4-8 weeks under the conditions of 27+/-1 ℃ and illumination for 13h every day and 3000Lx until complete quasi-transgenic plants are regenerated.

3. Obtaining sweet potato transgenic plant

Identification of transgenic plants: identification of the transgenic lines overexpressing IbNAC43 gene was performed using PCR detection. The genome DNA of the quasi-transgenic plant and the wild sweet potato plant is extracted by using a CTAB method, PCR detection is carried out on the genome DNA, and simultaneously, the pCAMBIA1300-IbNAC43 vector plasmid DNA is used as a positive control, water and the wild plant DNA are used as a negative control, and the PCR amplification is carried out by using a primer 5 and a primer 6.

Primer 5:5'-TCCTTCGCAAGACCCTTCCTC-3';

primer 6:5'-TACCGACAAGTGGGACAAGG-3'.

The amplified PCR product is electrophoretically separated in agarose gel of 1% (w/v), the PCR positive plant should have a specific 1156bp electrophoresis band, and the line number of the PCR positive plant is recorded, namely the transgenic line which is determined to over express the IbNAC43 gene.

The results of some experiments are shown in FIG. 1, and the results show that L4, L6 and L7 are positive plants transformed with IbNAC43 genes. The positive plants of the transgenic IbNAC43 gene obtained by the same method are L10, L12 and L14, namely F ₀ And (5) replacing transgenic plants.

Then, RNA of the whole strain of the wild type and all transgenic lines was extracted with TroZol kit, the expression level of IbNAC43 gene was detected with qRT-PCR, and the kit SYBR Premix Ex Taq was TaKaRa (Takara Shuzo Co., ltd., dalian) product (catalog number: RR 420), and primer 7 and primer 8 were used for analysis of the expression level.

Primer 7:5'-GAACCGTCCTGCAACAACAC-3';

primer 8:5'-GAGGATTCGATCTGGCCGTT-3'.

The results of the experiment are shown in FIG. 2, and the results show that the expression level of IbNAC43 gene in transgenic plants L4, L6, L7, L10, L12 and L14 is remarkably improved compared with that of Wild Type (WT). Transgenic plants L4, L6, L7, L10, L12 and L14 are sweet potato transgenic positive plants.

2. Identification of leaf curl of IbNAC43 transgenic sweet potato plants

1. Blade curl identification

And (3) propagating the wild plants of the sweet potatoes and the transgenic positive plants L4, L10 and L14 respectively by adopting a asexual propagation method to obtain a large number of plants.

The experiment was repeated three times, each time with the following steps:

(1) Sweet potato test tube plantlets (about 10cm long and containing stem tips) grown ex vivo for 4 weeks were planted in plastic flowerpots filled with artificial soil (made up of 1 part by volume vermiculite and 1 part by volume nutrient soil) and 4 plants per line.

(2) After the step (1) is completed, each basin is irrigated with 1/2 Hoagland nutrient solution for 2 weeks.

(3) After the step (2) is completed, each basin grows normally for 6 weeks (i.e. water is normally irrigated). After 6 weeks, the growth state of the sweet potato plants was observed, and the leaf curl index of the sweet potato plants was measured:

the natural distance (Ln) between the left and right edges of the blade was measured with a vernier caliper, and the maximum width (Lw) of the left and right edges thereof was measured with the blade stretched, with the rolling index LRI (%) = (Lw-Ln)/lw×100).

In addition, the determination of chlorophyll content and photosynthetic rate was performed on sweet potato plants. The chlorophyll content of leaves of sweet potato plants was examined by the method of the reference "Zhao Haibo, lin Qi, sun Xusheng, etc. Nitrogen phosphorus complex application on photosynthetic characteristics after flowers of Jimai 22 [ J ]. J. Wheat crop journal, 2009,029 (004): 663-667". And (3) measuring the photosynthetic rate of the sweet potato leaves by using an LI-6400 portable photosynthetic determinator, and selecting the plants growing for 8 weeks in the step (3) to measure the chlorophyll content and the photosynthetic rate of the leaves, wherein each plant is used for measuring 6 independent plants.

The growth state of sweet potato plants is shown in the A diagram of FIG. 3. The leaf curl index statistics of the sweet potato plants are shown in the graph B of FIG. 3, the chlorophyll content of the sweet potato plants are shown in the graph C of FIG. 3, and the photosynthesis rate is shown in the graph D of FIG. 3. The results showed that after 8 weeks of acclimated growth (from the planting in the pot in step (1)), the transgenic plants were significantly lower in plant height than the control wild type and the leaf curl index (LRI) of L4, L10 and L14 was 16.56, 19.18 and 15.92 times that of the control, respectively. Leaves of transgenic plants L4, L10 and L14 were significantly lower than wild type in both chlorophyll relative content and photosynthetic rate, resulting in a transgenic plant that was retarded in development due to lack of energy.

2. Observation of leaf tissue structure

(1) Scanning electron microscope observation

Changes in the morphology and structure of leaf cells typically cause changes in the leaf. In order to study the morphological changes of the leaves, samples for scanning electron microscope observation were prepared using transgenic plants L10 and L14 at week 8 of the above 1 and wild type plant leaves as materials. The pore morphology and pore size of the leaf were observed with a Hitachi S-3400N scanning electron microscope (Hitachi S3400N, tokyo, japan), and were observed and recorded.

The scanning electron microscope observation results are shown in a graph A of FIG. 4 (ad represents the front surface of the leaf, ab represents the back surface of the leaf), and the cells on the back surface of the leaf of the transgenic plant are found to have a lot of ravines compared with the front surface of the leaf, and the cells are compact and uneven in arrangement, while the cells on the epidermis of the leaf of the control wild plant are flat and tidy in arrangement on the front surface and the back surface.

(2) Transverse viewing of blades

Paraffin slicing results are shown in a diagram B of fig. 4, upper and lower epidermal cell statistics are shown in a diagram C of fig. 4, and the result shows that the number of the upper and lower epidermal cells of a wild type leaf per unit length is basically consistent, and the number of the lower epidermal cells of the leaf in a transgenic plant is obviously increased compared with that of the upper epidermis.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (7)

1. Any of the following applications of IbNAC43 protein or a substance that modulates the content or activity of said IbNAC43 protein:

   d1 Regulating and controlling plant leaf curl index;

   d2 Application of regulating chlorophyll content in plant leaves;

   d3 Regulating and controlling the photosynthetic rate of the plant leaves;

   d4 Regulating plant leaf morphology;

   d5 Regulating the relative ratio of the number of the upper and lower epidermal cells of the plant leaf;

   d6 Regulating plant leaf development;

   d7 Performing plant breeding applications;

   the IbNAC43 protein is the protein of A1, A2 or A3 as follows:

   a1, the amino acid sequence is protein of the amino acid sequence shown as SEQ ID No.2 in the sequence table;

   a2, carrying out substitution and/or deletion and/or addition of amino acid residues on an amino acid sequence shown in SEQ ID No.2 in a sequence table to obtain a protein which has more than 80% of identity with the protein shown in A1) and is related to plant leaf development;

   a3, N-terminal or/and C-terminal of A1) or A2).
2. 2. The use according to claim 1, characterized in that: the IbNAC43 protein is derived from sweet potato.
3. 3. Use according to claim 1 or 2, characterized in that: the substance regulating the content or activity of the IbNAC43 protein is a biological material related to the IbNAC43 protein according to claim 1; the biological material is any one of the following C1-C3:

   c1, a nucleic acid molecule encoding the IbNAC43 protein of claim 1;

   c2, nucleic acid molecules that increase expression of the protein;

   c3, expression cassette comprising a nucleic acid molecule as described in C1 or C2, recombinant vector, recombinant microorganism, transgenic plant cell line, transgenic plant tissue or transgenic plant organ.
4. 4. A use according to claim 3, characterized in that: the nucleic acid molecule C1 is a nucleic acid molecule with a coding sequence of a coding chain being SEQ ID No.1 in a sequence table.
5. 5. The use according to any one of claims 1 to 4, wherein the plant is any one of the following:

   c1 Dicotyledonous or monocotyledonous plants;

   c2 A plant of the order of the tubular flower,

   c3 A plant of the family Convolvulaceae,

   c4 A plant of the genus sweet potato,

   c5 Sweet potato.
6. 6. IbNAC43 protein as claimed in claim 1.
7. 7. The IbNAC43 protein-related biomaterial of claim 3 or 4.

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