Related gene LsPHR1 for regulating biomass of leaf lettuce and application of related gene LsPHR1

Abstract

The invention belongs to the field of plant genetic engineering, and particularly relates to a related gene LsPHR1 for regulating and controlling the biomass of leaf lettuce (lettuce) and application of the related gene LsPHR1. The amino acid sequence of the protein coded by the LsPHR1 gene is as shown in SEQ ID NO: 1, the CDS sequence is as shown in SEQ ID NO: 2, and the DNA full-length sequence is as shown in SEQ ID NO: 3. The invention also provides an application of the leaf lettuce biomass related gene LsPHR1 in increasing the biomass of leaf lettuce, and a method for increasing the biomass of leaf lettuce. According to the invention, related genes for controlling the biomass of leaf lettuce (lettuce) are excavated and studied, so that the biomass internal mechanism and regulatory network of leaf lettuce are explored, a theoretical basis is provided for cultivating high-biomass varieties, and the development of high-biomass lettuce varieties is facilitated.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant genetic engineering, and particularly relates to a biomass-related gene LsPHR1 for regulating Lactuca sativa and application thereof. BACKGROUND

[0002] Lactuca sativa L., commonly known as lettuce, is an annual or biennial herbaceous crop of the Lactuca genus of the Asteraceae family, and is fond of cool and cool environment. According to the 2024-2029 China Lettuce Industry Market Depth Research and Investment Strategy Forecast Report of Zhongyan Puhua Industry Research Institute, the global lettuce market size continues to grow in 2024; in recent years, the market size of China lettuce market has been expanding, showing strong growth momentum. Lettuce is an important leafy vegetable widely planted in the world due to its fresh and tender taste, rich nutrition, and high content of vitamins, minerals and dietary fiber. With the growth of consumer demand and the development of facility agriculture, the cultivation area of Lactuca sativa is expanding, and the requirements for its yield (biomass) and stress resistance are also increasing. Biomass is a core indicator for measuring the yield of Lactuca sativa, which is jointly regulated by genetics, environment and cultivation conditions, and the regulatory role of genes plays a decisive role. Therefore, excavating key genes related to biomass formation and cultivating high biomass through molecular breeding is the fundamental way to solve the above problems. Therefore, in the current research and practice, it is necessary to excavate and study the genes related to the regulation of lettuce biomass. SUMMARY

[0003] In order to solve the above problems existing in the prior art, the application provides a biomass-related gene LsPHR1 for regulating Lactuca sativa and application thereof.

[0004] The application provides a protein amino acid sequence encoded by the biomass-related gene LsPHR1 of Lactuca sativa, and the amino acid sequence is shown as SEQ ID NO: 1 in the sequence table.

[0005] The application also provides a CDS sequence of the biomass-related gene LsPHR1 of Lactuca sativa, and the nucleotide sequence of the CDS sequence is shown as SEQ ID NO: 2 in the sequence table.

[0006] The application also provides a DNA full-length sequence of the biomass-related gene LsPHR1 of Lactuca sativa, and the nucleotide sequence of the DNA full-length sequence is shown as SEQ ID NO: 3 in the sequence table.

[0007] The application also provides primers for amplifying the CDS sequence of the biomass-related gene LsPHR1 of Lactuca sativa, comprising:

[0008] Forward primer (SEQ ID NO: 4): 5'- ATGGAGACACAACACGCGTTAT-3';

[0009] Reverse primer (SEQ ID NO: 5): 5'- TTATGACGACGAGTCGTTTAAT-3'.

[0010] The application further provides a primer for amplifying the full-length sequence of the DNA of the leaf lettuce biomass-related gene LsPHR1, comprising:

[0011] Forward primer (SEQ ID NO: 6):

[0012] 5'- CTGTAGGTGTGATGGAGGAATGAT-3'.

[0013] Reverse primer (SEQ ID NO: 7):

[0014] 5'- ATACGATCGTCCGATCAACTGA-3'.

[0015] The application further provides an application of the leaf lettuce biomass-related gene LsPHR1 in increasing the biomass of leaf lettuce.

[0016] The application further provides a method for increasing the biomass of leaf lettuce, comprising: inhibiting the expression of LsPHR1 protein or reducing the content and / or activity of LsPHR1 protein by LsPHR1 gene knockout, or inhibiting the expression of LsPHR1 gene, or silencing LsPHR1 gene.

[0017] The application is helpful for exploring the internal mechanism and regulation network of the biomass, and provides a theoretical basis for cultivating a high-biomass variety, and is helpful for developing a high-biomass lettuce variety. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 It is a schematic diagram of the position of the LsPHR1 gene knockout target in the nucleotide sequence in Example 2.

[0019] Figure 2 It is a schematic diagram of the structure of the gene knockout vector LsPHR1-pZDK672 in Example 2.

[0020] Figure 3 It is a schematic diagram of the knockout target analysis of the LsPHR1 gene knockout plant in Example 2.

[0021] Figure 4 It is a photo of the phenotype of the LsPHR1 gene knockout plant in Example 2.

[0022] Figure 5 A histogram of leaf fresh weight of LsPHR1 gene knockout plants in Example 2.

[0023] Figure 6 A histogram of LsPHR1 gene expression amount of LsPHR1 gene knockout plants in Example 2. DETAILED DESCRIPTION

[0024] In order to make the technical solutions, objectives and advantages of the present application clearer, the present application will be further described in detail below through specific examples. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present application, and are not used to limit the present application.

[0025] In the present application, the term "biomass" refers to the total weight of the aboveground part of leaf lettuce (lettuce) after removing the roots, which can generally be considered as the leaf fresh weight of lettuce.

[0026] In the first aspect, the present application provides a protein amino acid sequence encoded by the LsPHR1 gene related to the biomass of leaf lettuce (lettuce), and the amino acid sequence is shown in SEQ ID NO: 1 in the sequence listing;

[0027] Or: the amino acid sequence has a homology of greater than or equal to 95% with SEQ ID NO: 1 in the sequence listing;

[0028] Or: on the basis of SEQ ID NO: 1 in the sequence listing, an amino acid sequence with the same function of the protein encoded by the LsPHR1 gene is obtained by substitution, and / or deletion, and / or addition of one or more amino acid residues.

[0029] The protein amino acid sequence (SEQ ID NO: 1) encoded by LsPHR1 is as follows:

[0030] METQHALSIQRSSPTQLSNHGTTRALSSSFPVHHTTLEEKHPKLLDSQLSSTNGTVGHIFSSSSGMSSDLHFSSISPNETCSKKAPFITQSSGVLRSTAFRQYMKENNTSSWSTDSLSDFLEYPESSPIEPTNLQPTPTENGSCDLPPEDFAKPNDWQDWADQLITEDDAATPNWNDILVDTEPKLELPVGGSSTKSMEVKKMVPASPGELCSPMTPSSCGGGSHSQSQNKPRMRWTPELHEAFVEAVNKLGGSERATPKGVLKQMKVEGLTIYHVKSHLQKYRTARYKPEPSSEGPSEKKPTSMQDLPSLDLKASLEMTEALRLQVEVQKRLHEQLEIQRNLQMRIEEQGKYLQMIFEKQCKFGIDNLKSSSSTHTPEKSETELLTNEISSSPVLTEPETERDPVKVGSPSSKVEVDPLESQPAKRAKLNDSSS.

[0031] In a second aspect, the present application provides a CDS sequence of a leaf lettuce (Lactuca sativa) biomass-related gene LsPHR1, wherein the nucleotide sequence of the CDS sequence is as set forth in SEQ ID NO: 2 in the Sequence Listing;

[0032] or: the nucleotide sequence of the CDS sequence has a homology of greater than or equal to 95% with SEQ ID NO: 2 in the Sequence Listing;

[0033] or: a nucleotide sequence encoding a protein having the same function as the protein encoded by the LsPHR1 gene, which is obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on SEQ ID NO: 2 in the Sequence Listing.

[0034] CDS sequence of LsPHR1 (SEQ ID NO: 2):

[0035]

[0036] In a third aspect, the present application provides a DNA full-length sequence of a leaf lettuce (Lactuca sativa) biomass-related gene LsPHR1, wherein the nucleotide sequence of the DNA full-length sequence is shown as SEQ ID NO: 3 in the Sequence Listing.

[0037] Or: the nucleotide sequence of the gene has a homology of greater than or equal to 95% with SEQ ID NO: 3 in the Sequence Listing.

[0038] Or: on the basis of SEQ ID NO: 3 in the Sequence Listing, a nucleotide sequence obtained by substitution, and / or deletion, and / or addition of one or more nucleotides, encodes a protein having the same function as the protein encoded by the LsPHR1 gene.

[0039] The DNA full-length sequence of LsPHR1 (SEQ ID NO: 3):

[0040]

[0041] In a fourth aspect, the present application provides a primer for amplifying the CDS sequence (SEQ ID NO: 2) of the leaf lettuce biomass-related gene LsPHR1, comprising:

[0042] a forward primer (SEQ ID NO: 4): 5'-ATGGAGACACAACACGCGTTAT-3';

[0043] a reverse primer (SEQ ID NO: 5): 5'-TTATGACGACGAGTCGTTTAAT-3'.

[0044] In a fifth aspect, the present application provides a PCR amplification primer for amplifying the full-length sequence (SEQ ID NO: 3) of the leaf lettuce biomass-related gene LsPHR1, comprising:

[0045] a forward primer (PHR-F) (SEQ ID NO: 6):

[0046] 5'-CTGTAGGTGTGATGGAGGAATGAT-3';

[0047] a reverse primer (PHR-R) (SEQ ID NO: 7):

[0048] 5'-ATACGATCGTCCGATCAACTGA-3'.

[0049] In a sixth aspect, the present application provides an application of the leaf lettuce biomass-related gene LsPHR1 in increasing the biomass (e.g., fresh weight increase) of leaf lettuce.

[0050] In a seventh aspect, the present application provides a method for increasing the biomass (e.g., fresh weight increase) of leaf lettuce, comprising:

[0051] inhibiting the expression of LsPHR1 protein or reducing the content and / or activity of LsPHR1 protein by knocking out the LsPHR1 gene, or inhibiting the expression of the LsPHR1 gene, or silencing the LsPHR1 gene.

[0052] According to the seventh aspect of the present application, the operation of knocking out the LsPHR1 gene comprises: designing a LsPHR1 gene knockout target, constructing a gene knockout vector (a frame vector can be selected as pZDK672 vector, or other gene knockout vectors can also be selected) of the LsPHR1 gene, and then transforming the gene knockout vector into leaf lettuce to obtain a transformed plant.

[0053] The LsPHR1 gene knockout target site sequence includes (SEQ ID NO: 8):

[0054] 5'-TTTGTGGATGATCCCCCCAC-3'.

[0055] The various reagents, materials, etc. used in the following examples, if not specifically stated, are products that can be obtained from commercial channels; the various tests, detection methods used in the following examples, if not specifically stated, are conventional tests, detection methods in the art, and can be obtained from textbooks, reference books, or academic journals.

[0056] Example 1

[0057] This example is used to illustrate the method of LsPHR1 gene and its PCR amplification.

[0058] The leaf lettuce (lettuce) biomass-related gene LsPHR1 is identified by the inventors through reverse genetics.

[0059] The amino acid sequence composition of the protein encoded by the above-mentioned gene LsPHR1 is shown in SEQ ID NO: 1 in the sequence listing; the nucleotide sequence composition of the CDS sequence of the above-mentioned gene LsPHR1 is shown in SEQ ID NO: 2 in the sequence listing; and the nucleotide sequence composition of the DNA full-length sequence of the above-mentioned gene LsPHR1 is shown in SEQ ID NO: 3 in the sequence listing.

[0060] The PCR amplification primer for amplifying the CDS sequence of the gene LsPHR1 includes:

[0061] Forward primer (SEQ ID NO: 4): 5'-ATGGAGACACAACACGCGTTAT-3';

[0062] Reverse primer (SEQ ID NO: 5): 5'-TTATGACGACGAGTCGTTTAAT-3'.

[0063] The PCR amplification primer for amplifying the DNA full-length sequence of the gene LsPHR1 includes:

[0064] Forward primer (PHR-F) (SEQ ID NO: 6):

[0065] 5'-CTGTAGGTGTGATGGAGGAATGAT-3';

[0066] Reverse primer (PHR-R) (SEQ ID NO: 7):

[0067] 5'-ATACGATCGTCCGATCAACTGA-3'.

[0068] The PCR amplification reaction for amplifying SEQ ID NO: 2, SEQ ID NO: 3 can use a conventional reaction system and a conventional PCR reaction procedure. For example, the PCR amplification reaction is performed using Novozyme 2xPhanta Flash Master Mix high-fidelity enzyme.

[0069] When amplifying SEQ ID NO: 2, RNA of the lettuce is first extracted, and then reverse transcribed into cDNA, and the PCR amplification is performed using the cDNA as a template; the PCR reaction system is: 2xPhanta Flash Master Mix 25 μL, forward primer 1 μL, reverse primer 1 μL, cDNA 100 ng, and ddH2O is supplemented to 50 μL.

[0070] The PCR reaction procedure for amplifying SEQ ID NO: 2 is: 98°C for 30s; 98°C for 10s, 56°C for 10s, 72°C for 15s, for a total of 34 cycles; 72°C for 1 min.

[0071] When amplifying SEQ ID NO: 3, the genomic DNA of the lettuce is first extracted, and then the PCR amplification is performed using the DNA as a template; the PCR reaction system is: 2xPhanta Flash Master Mix 25 μL, forward primer 1 μL, reverse primer 1 μL, DNA 100 ng, and ddH2O is supplemented to 50 μL.

[0072] The PCR reaction procedure for amplifying SEQ ID NO: 3 is: 98°C for 30s; 98°C for 10s, 56°C for 10s, 72°C for 40s, for a total of 34 cycles; 72°C for 1 min.

[0073] Example 2

[0074] This example is used to illustrate the obtaining of the lettuce mutant plant with LsPHR1 gene knockout and the analysis of the lettuce biomass.

[0075] I. Construction of gene knockout vector

[0076] 1. Design the target sequence of LsPHR1 gene:

[0077] The genomic sequence (SEQ ID NO: 3) of this gene is input into the CRISPOR (ucsc.edu) website, and the following sgRNA target (SEQ ID NO: 8) is designed: 5'-TTTGTGGATGATCCCCCCAC-3', the position of which on the genome is as shown in Figure 1As shown. This gene is located on chromosome 9 of lettuce, from 13169409 bp to 13172245 bp; i.e., the website Gene Report: Lsat_1_v5_gn_9_11321: Phytozome (doe.gov) The given value is L.sativa V8|Lsat_1_v5_gn_9_11321 Lsat_1_v8_lg_ 9:13169409..13172245 reverse.

[0078] 2. Based on the nucleotide sequences of the above target sites, design sgRNA sequences containing BsaI restriction enzyme sites and their reverse complementary sequences, and directly synthesize the following two sequences.

[0079] sgRNA-F (SEQ ID NO:9): 5'- TGCA TTTGTGGATGATCCCCCCAC-3';

[0080] sgRNA-R (SEQ ID NO:10): 5'- AAAC GTGGGGGGATCATCCACAAA-3'.

[0081] 3. Anneal the above sgRNA-F and sgRNA-R: Mix sgRNA-F and sgRNA-R in equal proportions; anneal them on a PCR instrument to pair them and form a double-stranded structure.

[0082] The annealing program is as follows: 95℃ for 5 min, 90℃ for 1 min, 80℃ for 1 min, 70℃ for 1 min, 60℃ for 1 min, 50℃ for 1 min, 40℃ for 1 min, 30℃ for 1 min, 20℃ for 1 min, and 10℃ for 1 min.

[0083] 4. The framework vector—gene editing vector pZDK672—was digested with Bsa I restriction enzyme to obtain a linearized vector.

[0084] The gene editing vector pZDK672 is described in "Establishment of an Efficient Genome Editing System in Lettuce Without Sacrificing Specificity", Wenbo Pan et al., Frontiers in Plant Science; URL: https: / / doi.org / 10.3389 / fpls.2022.930592.

[0085] The above enzyme digestion reaction system (30 μL) includes: 1 μL of BsaI endonuclease (NEB), 3 μL of 10×cutsmartbuffer, 2 μg of plasmid pZDK672, and ddH2O to a final volume of 30 μL.

[0086] The above enzyme digestion procedure is as follows: digest at 37℃ for 1~2 hours.

[0087] 5. The linearized vector and the double-stranded structure obtained in step 3 were ligated using T4 ligase (NEB T4 DNALigase (M0202)) to obtain the LsPHR1 gene knockout vector LsPHR1-pZDK672 targeting the above-mentioned target.

[0088] The above ligation reaction system (20 μL) includes: 2 μL of T4 DNA Ligase Buffer (10X), 5-12 μL of the double-stranded structure formed by linearized vector and annealed sgRNA-F and sgRNA-R in a molar ratio of 1:3, 1 μL of T4 DNA Ligase, and Nuclease-free water added to 20 μL.

[0089] The above connection reaction procedure: overnight connection at 16°C.

[0090] The structural diagram of the LsPHR1 gene knockout vector LsPHR1-pZDK672 is shown below. Figure 2 The vector LsPHR1-pZDK672 consists of the following components from upstream to downstream: 35S promoter, tRNA, sgRNA (i.e., double-stranded structure), tRNA, and Cas9.

[0091] II. Obtaining Genetically Modified Lettuce Plants

[0092] 1. The LsPHR1 gene knockout vector LsPHR1-pZDK672 was introduced into Agrobacterium tumefaciens EHA105 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) to obtain recombinant Agrobacterium. The recombinant Agrobacterium was then transformed into the lettuce variety LVYA (purchased from Jingyan Yinong (Beijing) Seed Industry Technology Co., Ltd.). The transformation procedure was performed according to the following literature: Zhang, H., X. Si, X. Ji, R. Fan, J. Liu et al., 2018 Genome editing of upstream openreading frames enables translational control in plants. Nat Biotechnol 36:894-898.

[0093] 2. Genomic DNA was extracted from the T0 generation plants obtained by transformation, and PCR reaction was performed to identify mutant plants; there were more than 3 mutant plants.

[0094] The primers used in the above PCR reaction are:

[0095] PHR-seqF (SEQ ID NO:11):5'-ATCAGATTTTCTTGAATACCCT-3';

[0096] PHR-seqR (SEQ ID NO:12): 5'-CATGATAAAATATAGCAAGGT-3'.

[0097] The above PCR reaction system (50 μL) includes:

[0098] 2×Taq PCR StarMix (GenStar Kangrun Biotechnology) 25μL, PHR-seqF primer 1μL, PHR-seqR primer 1μL, DNA 100ng, ddH2O to make up to 50μL.

[0099] The above PCR reaction program is as follows: 95℃ for 5 min; 95℃ for 20 s, 56℃ for 20 s, 72℃ for 30 s, for a total of 34 cycles; 72℃ for 5 min.

[0100] The PCR reaction products were then sequenced and analyzed, and the results are as follows.

[0101] 3. After planting the identified mutant plants (T0 generation) for about 4 months, harvest the seeds; continue planting to obtain T1 generation plants, perform the above PCR reaction to identify the mutant plants and harvest the seeds; continue planting to obtain T2 generation plants, perform the above PCR reaction to identify the mutant plants among them.

[0102] III. Results and Analysis

[0103] The wild type (WT) of the lettuce variety LVYA was used as a control.

[0104] 1. Knockout target analysis of LsPHR1 gene knockout plants

[0105] The sequencing results and analysis of the PCR reaction product in step 2 are as follows:

[0106] like Figure 3 As shown, the target site underwent the following two mutations.

[0107] The reverse complementary sequence of the target is: 5'-GTGGGGGGATCATCCACAAA-3'.

[0108] The sequence of WT is (SEQ ID NO:13):

[0109] 5'-GCTGGAACTTCCTGTGGGGGGATCATCCACAAAGTCAATG-3'.

[0110] Mutation 1 (LsPHRI-KO-1) (SEQ ID NO:14):

[0111] 5'-GCTGGAACTTCGGGGGATCATCCACAAAGTCAATG-3'.

[0112] Mutation 2 (LsPHRI-KO-2) (SEQ ID NO:15):

[0113] 5'-GCTGGAACTTCCTGTG T GGGGGATCATCCACAAAGTCAATG-3'.

[0114] It is evident that mutation 1 lacks CGTG compared to WT, while mutation 2 has one more T than WT.

[0115] 2. Phenotypic analysis of LsPHR1 gene knockout plants (T2 generation) at harvest time (70 days from seed sowing).

[0116] like Figure 4 As shown, the rosette leaves of the LsPHR1 gene knockout plants LsPHR1-KO-1 and LsPHR1-KO-2 have significantly larger biomass than those of the wild-type WT.

[0117] 3. Biomass (fresh weight of leaves) analysis of LsPHR1 gene knockout plants (70 days after sowing)

[0118] like Figure 5 As shown, the average biomass of WT was 0.21 kg, that of LsPHR1-KO-1 was 0.41 kg, and that of LsPHR1-KO-2 was 0.375 kg. It can be seen that the biomass of the LsPHR1 gene knockout plants LsPHR1-KO-1 and LsPHR1-KO-2 was much greater than that of wild-type WT, reaching about twice that of WT, and a significant difference was achieved between wild-type plants and gene knockout plants.

[0119] 4. Analysis of LsPHR1 gene expression level in LsPHR1 knockout plants (70 days after sowing)

[0120] The expression level of the LsPHR1 gene was detected as follows:

[0121] (1) Total RNA was extracted from the leaves of WT plants and gene knockout plants using the Total RNA Extraction Kit for Plants; the obtained total RNA was used as a template to reverse transcribe cDNA using the Novozymes HiScript III All-in-one RT SuperMix Perfectfor qPCR Reverse Transcription Kit.

[0122] (2) qRT-PCR experiments were performed using the Novizan Taq Pro Universal SYBR qPCR Master Mix kit with the above-mentioned cDNA as the template, and the relative expression level of LsPHR1 in transgenic lettuce was calculated.

[0123] The above qRT-PCR reaction used lettuce actin as an internal reference gene, and the primers were LsaActin-F / R.

[0124] LsaActin-F (SEQ ID NO:16): 5'-CTGGTGTGATGGTAGGTATGG-3';

[0125] LsaActin-R (SEQ ID NO:17): 5'-CTCGTTGTAGAAAGTGTGATGC-3'.

[0126] The primers for the above qRT-PCR reaction are:

[0127] PHR-qPCR-F (SEQ ID NO:18): 5'-ATTTACAACCCACTCCCACTG-3';

[0128] PHR-qPCR-R (SEQ ID NO:19): 5'-CCCAATCCTGCCAATCATTTG-3'.

[0129] The above qRT-PCR reaction system (20 μL) consists of: 10 μL of 2×PerfectStart Green qPCR Super Mix, 1 μL of Primer-F, 1 μL of Primer-R, 200 ng of cDNA, and ddH2O to a final volume of 20 μL.

[0130] The above qRT-PCR reaction program is as follows: 95℃ for 3 min; 95℃ for 10 s, 60℃ for 30 s, 40 cycles; 95℃ for 10 s, 60℃ for 1 min; 95℃ for 15 s.

[0131] (3) After the qRT-PCR reaction is complete, use 2 −ΔΔCtThe relative expression level of the LsPHR1 gene in WT plants and LsPHR1 gene knockout plants was calculated using a method.

[0132] like Figure 6 As shown, the average relative expression level of LsPHR1 in wild-type WT was 0.156, in LsPHR1-KO-1 it was 0.068, and in LsPHR1-KO-2 it was 0.055. It is evident that the relative expression level of LsPHR1 in the LsPHR1 knockout plants LsPHR1-KO-1 and LsPHR1-KO-2 was significantly lower than that in wild-type WT, decreasing to approximately half that of WT, indicating a significant difference between the wild-type and knockout plants.

[0133] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The amino acid sequence of the protein encoded by the biomass-related gene LsPHR1 in leaf lettuce, the amino acid sequence composition of which is shown in SEQ ID NO:1 in the sequence listing; Alternatively: the amino acid sequence composition has greater than or equal to 95% homology with SEQ ID NO:1 in the sequence listing; Alternatively: Based on SEQ ID NO:1 in the sequence listing, an amino acid sequence having the same function as the protein encoded by the LsPHR1 gene is obtained by substitution, deletion, and / or addition of one or more amino acid residues.

2. The CDS sequence of LsPHR1, a biomass-related gene in leaf lettuce, wherein the nucleotide sequence composition of the CDS sequence is shown in SEQ ID NO:2 in the sequence listing; Alternatively: The nucleotide sequence composition of the CDS sequence has greater than or equal to 95% homology with SEQ ID NO:2 in the sequence listing; Alternatively: A nucleotide sequence encoding a protein having the same function as the protein encoded by the LsPHR1 gene, obtained by substitution, deletion, and / or addition of one or more nucleotides based on SEQ ID NO:2 in the sequence listing.

3. The full-length DNA sequence of LsPHR1, a biomass-related gene in leaf lettuce, wherein the nucleotide sequence composition of the full-length DNA sequence is shown in SEQ ID NO:3 in the sequence listing; Alternatively: The nucleotide sequence composition of the gene has greater than or equal to 95% homology with SEQ ID NO:3 in the sequence listing; Alternatively: A nucleotide sequence encoding a protein having the same function as the protein encoded by the LsPHR1 gene, obtained by substitution, deletion, and / or addition of one or more nucleotides based on SEQ ID NO:3 in the sequence listing.

4. Primers for amplifying the CDS sequence of the leaf lettuce biomass-related gene LsPHR1 as described in claim 2, comprising: Forward primer (SEQ ID NO:4): 5'-ATGGAGACACAACACGCGTTAT-3'; Reverse primer (SEQ ID NO:5): 5'-TTATGACGACGAGTCGTTTAAT-3'.

5. Primers for amplifying the full-length DNA sequence of the leaf lettuce biomass-related gene LsPHR1 as described in claim 3, comprising: Forward primer (SEQ ID NO:6): 5'-CTGTAGGTGTGATGGAGGAATGAT-3'; Reverse primer (SEQ ID NO:7): 5'-ATACGATCGTCCGATCAACTGA-3'.

6. Application of LsPHR1, a biomass-related gene in leaf lettuce, in increasing the biomass of leaf lettuce.

7. A method for increasing the biomass of leaf lettuce, comprising: Operations that inhibit LsPHR1 protein expression or reduce LsPHR1 protein content and / or activity by knocking out, suppressing, or silencing the LsPHR1 gene.

8. The method according to claim 7, characterized in that: The LsPHR1 gene knockout operation includes: designing an LsPHR1 gene knockout target, constructing a gene knockout vector for the LsPHR1 gene, and then transferring the gene knockout vector into leaf lettuce to obtain transformed plants.

9. The method according to claim 8, characterized in that: The LsPHR1 gene knockout target sequence includes (SEQ ID NO:8): 5'-TTTGTGGATGATCCCCCCAC-3'.

10. The method according to claim 8 or 9, characterized in that: The framework vector for the gene knockout vector is the pZDK672 vector.

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