Gossypium hirsutum leaf roll regulation gene ghclg and application thereof

Abstract

The application discloses a Gossypium hirsutum l. leaf roll regulation gene GHCLG and application thereof. The nucleotide sequence of the gene in a normal broadleaf Gossypium hirsutum variety TM-1 is shown in SEQ ID NO. 1, and the nucleotide sequence of the gene in a Gossypium hirsutum leaf roll mutant T582 is shown in SEQ ID NO. 3. The gene cloned by the application has a direct connection with cotton leaf shape. Transgenic research is carried out on normal broadleaf Gossypium hirsutum 668 by using a constructed plant gene editing vector, and the result shows that knocking out the GHCLG gene leads to cotton leaf shape upwarping and inrolling, so that the gene has great application potential in improving plant leaf shape and plant morphology, improving plant photosynthetic efficiency and resisting pests and the like.

Description

Technical Field

[0001] This invention relates to a gene GHCLG that regulates leaf curling in upland cotton. This gene sequence was obtained from upland cotton and belongs to the field of biotechnology applications. Background Technology

[0002] Leaves are the primary site of photosynthesis in plants. Studying the molecular mechanisms of leaf development provides a theoretical basis for improving plant photosynthetic efficiency. Leaf development mainly involves several stages, including the initiation of leaf primordia development, the establishment of polarity, leaf expansion, and maturation. During plant vegetative growth, leaves continuously emerge from the lateral aspect of the apical meristem (SAM). The shape of the leaf is determined at the initiation stage of leaf primordia development. The development from small protuberances on the SAM into mature leaves is accompanied by the inhibition of class-1 KNOTTED-like homeobox (KNOX) expression and local auxin accumulation during leaf primordia initiation (Long et al., 1996).

[0003] Leaf shape is an important aspect of leaf morphogenesis, regulated by complex interactions between genetic networks (Ha et al., 2010). Any defect or disruption in axillary-abaxial polarity development leads to abnormal leaf shape. Appropriate leaf curling contributes to a more upright plant form, thereby increasing light capture and reducing transpiration loss, which in turn improves photosynthetic efficiency and resistance to environmental stresses (Lang et al., 2004; Wu et al., 2010; Li et al., 2017). Simultaneously, upward leaf curling also reduces the living space of pests on the underside of leaves, such as whiteflies and winged whiteflies, thus playing a role in pest control. Therefore, leaf curling is considered an important agronomic trait in breeding and has been widely applied to high-yielding rice varieties (Zhu et al., 2001; Chen et al., 2010; Li et al., 2017). Cotton (Gossypium spp.) is an important economic and oilseed crop worldwide, cultivated in over 100 countries and regions. Factors such as leaf photosynthetic efficiency and pest infestation affect cotton yield and fiber quality. Although several genetic regulators of leaf curling, such as RLD1, SFL1, and OsZHD1 (Juarez et al., 2004; Xu et al., 2014; Alamin et al., 2017), have not yet been found in cotton to be associated with the leaf curling phenotype. Therefore, studying the genes controlling leaf curling in upland cotton is helpful in breeding cotton varieties with high photosynthetic efficiency or ideal plant architecture. Summary of the Invention

[0004] The purpose of this invention is to provide a cotton curling leaf gene (GHCLG) and its applications. In a first aspect, this invention provides the sequences of the upland cotton curling leaf regulatory gene GHCLG in the upland cotton genetic standard line TM-1 and the curling leaf mutant T582; wherein, the nucleotide sequence of this gene in upland cotton (G. hirsutum) TM-1 is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; and the nucleotide sequence of this gene in upland cotton (G. hirsutum) T582 is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4.

[0005] SEQ ID NO.1:

[0006]

[0007] The present invention describes the cotton GHCLG gene. A pair of GHCLG gene-specific primers were designed to detect the gene in upland cotton TM-1 and T582. The results showed that the gene had a 24bp base difference in TM-1 (broadleaf type) and T582 (curled leaf type), which resulted in a 6-amino acid difference after translation. This indicates that the amino acid difference of the gene may be the key to leaf curling.

[0008] In a second aspect, this invention provides the application of the upland cotton leaf-curling regulatory gene GHCLG (shown in SEQ ID NO.2) in regulating leaf shape. The application utilizes genetic engineering techniques to knock out the upland cotton leaf-curling regulatory gene GHCLG (shown in SEQ ID NO.2), causing the leaf edges to curl upwards, thus altering the plant's leaf shape. Leaf curling contributes to a more upright plant form, enhances the plant's resistance to lodging, reduces leaf water transpiration loss, and also compresses the living space of pests on the underside of the leaves, thus playing a role in the upland cotton's resistance to pests. Leaf curling is an important agronomic trait and is currently being widely applied in rice cultivation.

[0009] Furthermore, based on the sequence of the GHCLG gene, a gene editing vector containing two sgRNA fragments was designed: sgRNA1 is located at 21–41 bp of the GHCLG gene, and sgRNA2 is located at 502–522 bp of the GHCLG gene. Gene-edited upland cotton material was obtained by infecting the cotton high-efficiency regeneration receptor 668 with Agrobacterium-mediated transformation.

[0010] The primers used include SEQ ID NO.7 to NO.12:

[0011] SEQ ID NO.7: pRGEB32-7 s 5'-AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG-3';

[0012] SEQ ID NO.8: inf pRGEB32-7 s 5'-AAGCATCAGATGGGCAAACAAA-3';

[0013] SEQ ID NO.9: GHCLG as 5'-CATGGCATACAATCGATGCTtgcaccagccgggaat-3';

[0014] SEQ ID NO.10: GHCLG 2s 5'-AGCATCGATTGTATGCCATGgttttagagctagaaata-3';

[0015] SEQ ID NO.11: GHCLG 2as 5'-ATATTGTTTGCTAGCAACAGtgcaccagccgggaat-3';

[0016] SEQ ID NO. 12: inf GHCLG as 5'-ttctagctctaaaacATATTGTTTGCTAGCAACAG-3'.

[0017] In a third aspect, this invention provides a method for cultivating a new upland cotton germplasm. By using genetic engineering techniques to knock out the upland cotton leaf curling regulatory gene GHCLG shown in SEQ ID NO.2, a new upland cotton germplasm is obtained. The new germplasm has leaves with upturned and curled edges, and can be promoted and applied in production.

[0018] The beneficial effects of this invention are as follows: This invention conducts transgenic research on upland cotton 668 using a constructed plant gene editing vector. The results show that after knocking out the GHCLG gene, the cotton leaf shape changed significantly, with the leaf edges curling upwards to form rolled leaves, making the entire plant more upright, which is beneficial to the field production of upland cotton and provides technical support for the promotion and application of the leaf-curling gene in production. Attached Figure Description

[0019] Figure 1 A comparison of leaf morphology between the parental lines TM-1 and T582. Scale bar: 5cm.

[0020] Figure 2 Images of the apical meristems of the parents TM-1 and T582 were obtained using scanning electron microscopy (SEM). Magnification was 200x, and the scale bar was 100 μm.

[0021] Figure 3 This is an amino acid sequence alignment of the GHCLG gene from the parents TM-1 and T582. The black dots indicate the amino acid differences.

[0022] Figure 4 Leaf morphology comparison between two GHCLG gene knockout cotton lines (CR#13 and CR#14) and the control group 668 plants. The leaf shape shown in the cut end exhibits a significant change. Scale bar: 3 cm.

[0023] Figure 5 To examine the apical meristem of GHCLG gene knockout cotton plants and the control group 668 plants using scanning electron microscopy, leaf shape changes were observed at the leaf primordia stage. The leaf tips of the transgenic recipient 668 plants grew upwards, while the tips of young leaves of CR#13 and CR#14 showed bending. Magnification was 200x, scale bar 100 μm.

[0024] Figure 6To examine the editing status of the GHCLG gene in GHCLG gene knockout cotton plants, CR#13 and CR#14 were GHCLG gene knockout plants, and 668 was the control group. sgRNA1 editing resulted in a one-base insertion in both CR#13 and CR#14, leading to subsequent amino acid translation mismatches and achieving the gene knockout effect. sgRNA2 editing was unsuccessful and therefore not shown. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for illustration and explanation of the present invention and are not intended to limit the present invention. Unless otherwise specified, the following embodiments are all conventional methods. Unless otherwise specified, the reagents and materials mentioned are all commercially available.

[0026] Example 1

[0027] Obtaining the full-length sequence of the cotton GHCLG gene.

[0028] Leaf samples were taken from both parents (TM-1 and T582), RNA was extracted, and cDNA was obtained by reverse transcription. Specific primers were designed (as shown in Table 1), and a fragment of approximately 1,600 bp was amplified from the cDNA template by PCR. This fragment was ligated into a T vector, transformed into DH5α, and after 12 hours, single colonies were picked for culture and sequencing. The returned sequences were repeatedly compared to reveal differences in the GHCLG gene sequences of both parents, with the main differences being in 6 amino acids. Figure 2 The results indicate that the GHCLG amino acid sequence in leaf roll T582 is mutated.

[0029] Table 1. Specific primers for PCR amplification

[0030]

[0031] Example 2

[0032] Functional verification of cotton GHCLG gene

[0033] pRGEB32 is a plant gene editing vector with the cotton U6 promoter. First, a sequence containing the adapter fragment is amplified using a template and ligated into the intermediate vector pDONR223. Then, it is recombined into the plasmid pRGEB32, which is digested by the restriction endonuclease BSTb1. The plasmid is then transformed into *E. coli*, spread onto resistant culture dishes, and incubated overnight at 37°C. Single colonies are then picked and shaken for identification.

[0034] The GHCLG gene editing vector was designed and constructed using the gene sequence SEQ NO ID.1 of this invention. The specific process is as follows:

[0035] First, referring to the CRISPR-P website http: / / cbi.hzau.edu.cn / crispr / , we identified editing sites with low off-target rates on the GHCLG gene. This project designed two editing regions: the sgRNA1 fragment located at 21–41 bp of the GHCLG gene; and the sgRNA2 fragment located at 502–522 bp of the GHCLG gene. Design primers pRGEB32-7 s:5'-AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG-3'(SEQ ID NO.7), inf pRGEB32-7 s:5'-AAGCATCAGATGGGCAAACAAA-3'(SEQ ID NO.8), GHCLG as:5'-CATGGCATACAATCGATGCTtgcaccagccgggaat-3'(SEQ ID NO.9), GHCLG 2s: 5'-AGCATCGATTGTATGCCATGgttttagagctagaaata-3' (SEQ ID NO. 10), GHCLG 2as: 5'-ATATTGTTTGCTAGCAACAGtgcaccagccgggaat-3' (SEQ ID NO. 11), inf GHCLG as:5'-ttctagctctaaaacATATTGTTTGCTAGCAACAG-3' (SEQ ID NO. 12). PCR was performed using primers GHCLG as with pGTR4 vector plasmid as template. The first PCR amplified sgRNA1 and sgRNA2 using pRGEB32-7S and GHCLG as, respectively; GHCLG 2S and GHCLG 2As were used for amplification. The second PCR used the product of the first PCR as template, amplified with primers IF pRGEB32-7S and IF GHCLG as, aiming to ligate sgRNA1 and sgRNA2 into a single fragment. A band of approximately 350 bp was amplified, gel-cleaved, and ligated into BSAI-digested pRGEB32 using recombination ligation. Recombination was performed according to the kit instructions, and the recombinant plasmid was transformed into competent DH5α cells of *E. coli*, cultured overnight, and then picked for detection.

[0036] GHCLG gene-edited cotton plants were obtained through Agrobacterium-mediated gene transformation.

[0037] 1) Remove the hulls from the cottonseeds, sterilize by shaking with hydrogen peroxide for 30 minutes, and wash twice with sterile water;

[0038] 2) Place 6 seeds in each bottle into etiolation seedling culture medium, incubate at 28 degrees Celsius in the dark, and after 24 hours, support the seedlings and carefully remove the seed coat;

[0039] 3) After 5-6 days, when the stem has grown to about 10cm, begin preparations for infection;

[0040] 4) Shake the bacteria in advance, collect 2ml of bacterial suspension (OD=0.5-0.6) on the same day, adjust the OD=0.1 with 10ml of MGL resuspension, and add 25μL of AS;

[0041] 5) Remove the etiolated seedlings and place them on sterile filter paper in a petri dish. Cut off the roots and cotyledons, leaving only the stems. Cut the stems into 0.5cm sections with a smooth cut surface.

[0042] 6) Immerse the cut stems in the MGL resuspended bacterial solution, ensuring the solution covers the stems, and leave for 10 minutes; pour off the bacterial solution and place the stems on clean filter paper to air dry; transfer the stems neatly, without touching each other, to a co-culture dish covered with clean filter paper and incubate in the dark for 36-48 hours.

[0043] 7) After co-culture, remove the stems and place them separately on 2-4D medium (with appropriate antibiotics and termethin) and culture normally; subculture once every 3 weeks until embryogenic callus is produced, then transfer the embryogenic callus tissue to differentiation medium and continue to culture until it grows into seedlings.

[0044] 8) When the seedlings have taken root and grown to the size of the mouth of the culture bottle, add water and open the lid to harden the seedlings for 1-2 days. When the seedlings have grown a large number of white new roots in sterile water, they are transferred to a greenhouse or transplanted to the field.

[0045] The following methods were used to detect gene editing:

[0046] DNA was extracted from the tender leaves of the obtained GHCLG gene-edited cotton seedlings. Primers were designed and identified based on the gene editing target sites: CCT-CRSP-F1: 5'-GACTAATTCAAGTTCCACTTCCCCC-3' (SEQ ID NO. 13), CCT-CRSP-R1: 5'-ATTGGTTACGCAGAGAGCAACGAGT-3' (SEQ ID NO. 14), CCT-CRSP-F2: 5'-GTTCCACTTCCCCCCATTTC-3' (SEQ ID NO. 15), and CCT-CRSP-R2: 5'-TTGATTGGTTACGCAGAGAG-3' (SEQ ID NO. 16). Library construction and sequencing were performed using the Hi-Tom sequencing method. The sequencing results were compared, and the gene editing status of the two GHCLG gene knockout plants (CR#13 and CR#14) and the control group (668 plants) is as follows: Figure 6It can be seen that sgRNA1 editing resulted in the insertion of one base in each of CR#13 and CR#14, leading to subsequent amino acid translation mismatches and achieving the gene knockout effect. sgRNA2 editing was unsuccessful and therefore not shown. A comparison of leaf shapes between the two GHCLG gene knockout plants (CR#13 and CR#14) and the control group 668 plants is shown below. Figure 4 As shown, the leaf shape comparison of the parental lines TM-1 and T582 is as follows: Figure 1 As shown, the leaf shape of the two GHCLG gene knockout plants (CR#13 and CR#14) changed significantly, with the leaf edges curling upwards to form rolled leaves, similar to the leaf shape of the rolled-leaf mutant T582. In contrast, the leaf shape of the control group 668 plants was similar to that of TM-1. This indicates that the GHCLG gene can regulate the leaf shape of upland cotton, and knocking out the expression of the GHCLG gene will produce rolled leaves.

[0047] Example 3

[0048] Scanning electron microscopy observation of shoot tips of GHCLG gene-edited plants and parent plants

[0049] Apical meristems were collected from two GHCLG gene knockout plants (CR#13 and CR#14), the transgenic recipient 668 plant, and the parents TM-1 and T582. The samples were fixed in 2.5 vol% glutaraldehyde fixative, washed three times with PBS for 15 min each time, fixed with 1 vol% osmium tetroxide for 2 h, and then washed three times with PBS buffer for 15 min each time. The samples were dehydrated by treating with 50 vol%, 70 vol%, 90 vol%, and 100% ethanol for 15 min each. The tissues were fixed on an aluminum stage using special double-sided tape, sputtered with gold, and then the development of young leaves was observed by scanning electron microscopy. The results are shown in the figure below. Figure 2 and Figure 5 It was found that the leaves of the two GHCLG gene knockout lines began to bend inward at the leaf primordia stage, similar to the structure of the leaf-curling mutant T582. The leaves of the control group did not bend upward as they grew, similar to the structure of TM-1. This further illustrates that the GHCLG gene can regulate the leaf shape of upland cotton, and inhibiting the expression of the GHCLG gene will result in leaf curling.

[0050] In summary, the upland cotton leaf curling gene GHCLG discovered in this invention can regulate the production of leaf curling in upland cotton, and therefore has great application potential in the breeding of new upland cotton varieties, improving photosynthetic efficiency and resistance to environmental stress.

[0051] The above description is a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. It should be noted that any modifications, improvements, and equivalent substitutions made by those skilled in the art within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A gene for the leaf shape of upland cotton GHCLG Its application in inducing leaf curling in upland cotton plants is characterized by... The nucleotide sequence of this gene is shown in SEQ ID NO.1, and the specific application is as follows: By knocking out or suppressing the upland cotton leaf shape gene in upland cotton plants GHCLG The expression of this allows upland cotton plants to curl their leaves.

2. The application according to claim 1, characterized in that, Knock out or suppress the upland cotton leaf shape gene in upland cotton plants GHCLG The primers used for expression include SEQ ID NO.7~NO.12: SEQ ID NO.7: pRGEB32-7 s 5'-AAGCATCAGATGGGCAAACAAAGCACCAGTGGTCTAG-3'; SEQ ID NO.8: inf pRGEB32-7 s 5'-AAGCATCAGATGGGCAAACAAA-3'; SEQ ID NO.9: GHCLG as 5'-CATGGCATACAATCGATGCTtgcaccagccgggaat-3'; SEQ ID NO.10: GHCLG 2 s 5'-AGCATCGATTGTATGCCATGgttttagagctagaaata-3'; SEQ ID NO.11: GHCLG 2 as 5'-ATATTGTTTGCTAGCAACAGtgcaccagccgggaat-3'; SEQ ID NO. 12: inf GHCLG as 5'-ttctagctctaaaacATATTGTTTGCTAGCAACAG-3'.

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