WO2021189832A1 - ZmSBP12基因在调控玉米抗旱性、株高及穗位高中的用途 - Google Patents

ZmSBP12基因在调控玉米抗旱性、株高及穗位高中的用途 Download PDF

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WO2021189832A1
WO2021189832A1 PCT/CN2020/122356 CN2020122356W WO2021189832A1 WO 2021189832 A1 WO2021189832 A1 WO 2021189832A1 CN 2020122356 W CN2020122356 W CN 2020122356W WO 2021189832 A1 WO2021189832 A1 WO 2021189832A1
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zmsbp12
gene
plant
corn
height
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王海洋
谢钰容
赵斌斌
王宝宝
赵永平
孔德鑫
李全权
李耀耀
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华南农业大学
中国农业科学院生物技术研究所
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Definitions

  • the present invention relates to the new use of ZmSBP12 gene, in particular to the new use of ZmSBP12 gene in regulating the drought resistance, plant height and ear height of corn, and belongs to the application field of ZmSBP12 gene in corn breeding.
  • Drought is an important environmental factor affecting the stable production of corn in China and the world.
  • the arid and semi-arid regions in the world account for about 34.9% of the earth’s land area and 42.9% of the cultivated land area. 60% of food loss.
  • Drought is an important environmental factor that affects the stable production of corn in China and the world. Nearly 70% of corn in China is distributed in the northeast, north, southwest, and northwest hilly drylands or plain drylands that rely on natural rainfall. Jilin province, the largest planted area, generally reduces production in dry years. Over 25%, the yield will be reduced by 30% to 35% in severe drought years, and the harvest will be almost impossible in some areas in severe drought years.
  • ZmSBP12 gene is a maize SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor regulated by miR156.
  • SPL SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE
  • the ZmSPL12 gene has 4 exons and 3 introns. Its main functional regions include miR156 regulatory site (CATGCTCTCTCTTCTGTCA) and SBP domain.
  • the drought resistance, plant height, and ear height of maize are often controlled by many minor quantitative trait loci (QTL).
  • QTL quantitative trait loci
  • maize breeding still needs to accumulate these minor QTLs to improve drought resistance, reduce plant height and ear position. High, low selection efficiency. Therefore, it has important theoretical and practical significance to dig out the main genes that control drought resistance, plant height and ear height in maize, and analyze its genetic regulatory network.
  • the main purpose of the present invention is to provide the use of ZmSBP12 gene in the regulation of drought resistance, plant height or ear height of corn;
  • the maize mutant plants have such traits as increased drought resistance, decreased plant height and ear height.
  • Overexpression of ZmSBP12 gene leads to increased drought resistance and decreased plant height and ear height, indicating that ZmSBP12 gene plays a vital role in drought resistance and plant type (plant height) of corn.
  • the drought resistance, plant height and ear height of maize can be regulated; for example, by increasing the expression abundance of ZmSBP12 in maize, it can improve the drought resistance of maize and reduce the plant height of maize. Or ear height; on the contrary, reducing the abundance of ZmSBP12 gene expression in corn can reduce the drought resistance of corn and increase the height of corn plant or ear height.
  • the present invention achieves the effects of improving the drought resistance of corn and reducing the plant height and ear height of corn by increasing the expression abundance of the ZmSBP12 gene. Therefore, it all involves the regulation of the mRNA and protein abundance of the ZmSBP12 gene to improve the drought resistance of corn and reduce the corn
  • the methods of plant height and ear height should fall within the protection scope of the present invention.
  • the methods described here should include all methods of artificial synthesis and design of nucleotides or proteins and the use of unreported natural mutations.
  • the expression vector enables the ZmSBP12 gene to be overexpressed or overexpressed in maize, thereby increasing the drought resistance of maize or reducing the plant height or ear height of maize, and then selecting and breeding transgenic plants with strong stress resistance (drought resistance) and lodging resistance Or through gene editing technology or gene knockout technology, the ZmSBP12 gene in the plant is subjected to functional defect mutations to obtain transgenic plants that are not drought-resistant and have increased plant height and ear height.
  • the present invention also relates to the application of the ZmSBP12 gene in maize breeding; including in the breeding process of maize inbred lines and hybrids; specifically, the ZmSBP12 gene can be overexpressed in one of the maize hybrid parents to reduce the plant height of maize , The ear position is high, and the effect of improving the drought resistance of corn.
  • the present invention provides a method for improving drought resistance in corn, which includes: (1) constructing a recombinant plant expression vector containing the ZmSBP12 gene; (2) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; 3) Overexpression of ZmSBP12 gene in plant tissues or cells.
  • the present invention also provides a method for improving the lodging resistance of corn, which includes: (1) constructing a recombinant plant expression vector containing the ZmSBP12 gene; (2) transforming the constructed recombinant plant expression vector into plant tissues or plant cells; 3) Overexpression of ZmSBP12 gene in plant tissues or cells.
  • the improvement of drought resistance and reduction of plant height and ear height of corn in the present invention are embodied in the enhanced drought resistance and reduction of plant height and ear height of the transgenic plant and the wild type under the same conditions.
  • the ZmSBP12 gene can be operably connected with the expression control element to obtain a recombinant plant expression vector that can express the coding gene in plants;
  • the recombinant plant expression vector can be composed of the 5′-end non-coding region, SEQ ID No. 2
  • the 3'non-coding region may include terminator sequences, mRNA cleavage sequences and the like. Suitable terminator sequences can be taken from the Ti-plasmid of Agrobacterium tumefaciens, such as the terminator regions of octopine synthase and nopaline synthase.
  • the recombinant plant expression vector may also contain a selectable marker gene for selecting transformed cells.
  • Selectable marker genes are used to select transformed cells or tissues. Marker genes include genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker genes also include phenotypic markers, such as ⁇ -galactosidase and fluorescent protein.
  • those skilled in the art can optimize the polynucleotide shown in SEQ ID No. 2 to enhance the expression efficiency in plants.
  • the preferred codons of the target plant can be used to optimize the synthesis of polynucleotides to enhance the expression efficiency in the target plant; or the polynucleotide shown in SEQ ID No. 2 can be site-modified to obtain a modified variant
  • the polynucleotide sequence of the variant is shown in SEQ ID No.6.
  • the invention also provides a recombinant plant expression vector containing the ZmSBP12 gene and a host cell containing the recombinant plant expression vector.
  • the coding amino acid sequence of the ZmSBP12 gene in the present invention is shown in SEQ ID No. 1. Due to the particularity of the amino acid sequence, any fragment or variant of the peptide protein containing the amino acid sequence shown in SEQ ID NO.1, such as its conservative variant, biologically active fragment or derivative, as long as the fragment or peptide protein of the peptide protein
  • the homology between the variant and the aforementioned amino acid sequence is more than 90%, and all belong to the protection scope of the present invention.
  • the specific changes may include deletions, insertions or substitutions of amino acids in the amino acid sequence; wherein, for conservative changes in the variants, the substituted amino acids have similar structures or chemical properties to the original amino acids, such as replacing isoforms with leucine. Leucine, variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • the nucleotide sequence of the ZmSBP12 gene is shown in SEQ ID No.2. Due to the particularity of the nucleotide sequence, any variant of the polynucleotide shown in SEQ ID NO. 2 as long as it has more than 90% homology with the polynucleotide falls within the protection scope of the present invention.
  • the variant of the polynucleotide refers to a polynucleotide sequence with one or more nucleotide changes.
  • the variant of this polynucleotide can be a raw variant or a non-bearing variant, including substitution variants, deletion variants and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide. It may be a substitution, deletion or insertion of a polynucleotide, but it will not substantially change the function of the peptide protein it encodes.
  • the present invention also relates to specific amplification primers for detecting the expression of ZmSBP12 gene, the sequences of which are shown in SEQ ID No. 3 and SEQ ID No. 4, respectively;
  • Upstream primer 5’-AGCTCATCTGACTTAAAGCCCC-3’
  • the specific amplification primers are used to amplify corn, and the product fragment is 155bp, and the product sequence is shown in SEQ ID No. 5.
  • the present invention achieves the effects of improving the drought resistance of maize, reducing the plant height and ear height of maize, and can be applied to assist in the selection of new maize varieties with drought resistance and lodging resistance, and can also be applied to excellent maize varieties. Breeding of cross lines and hybrids.
  • polynucleotide or “nucleotide” means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally-occurring nucleotides.
  • oligonucleotide analogs which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, etc.) .
  • PNA peptide nucleic acid
  • DNA analogs used in antisense technology phosphorothioates, phosphoramidates, etc.
  • a specific nucleic acid sequence also implicitly encompasses conservatively modified variants (including but not limited to degenerate codon substitutions) and complementary sequences as well as explicitly specified sequences.
  • degenerate codon substitution can be achieved by generating a sequence in which one or more selected (or all) codons are substituted with mixed bases and/or deoxyinosine residues at position 3 (Batzer et al. , Nucleic Acid Res.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • polypeptide polypeptide
  • the term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally encoded amino acids.
  • the term encompasses amino acid chains of any length, including full-length proteins (ie, antigens) in which the amino acid residues are linked via covalent peptide bonds.
  • variant means a substantially similar sequence.
  • a variant includes a deletion, insertion, or/and substitution of one or more nucleotides at one or more positions in the natural polynucleotide.
  • conservative variants include those that do not change the encoded amino acid sequence due to the degeneracy of the genetic code. Such naturally occurring variants can be identified by existing molecular biology techniques.
  • Variant polynucleotides also include polynucleotides of synthetic origin, for example, polynucleotide variants that still encode the amino acid sequence shown in SEQ ID No. 1 obtained by site-directed mutagenesis or through recombination methods (such as DNA shuffling) .
  • stringent hybridization conditions in the present invention means low ionic strength and high temperature conditions known in the art. Generally, under stringent conditions, the detectable degree of hybridization between a probe and its target sequence is higher than that of hybridization with other sequences (for example, at least 2 times more than the background. Stringent hybridization conditions are sequence-dependent, and in different environments The conditions will be different. Longer sequences hybridize specifically at higher temperatures. By controlling the stringency of hybridization or washing conditions, the target sequence that is 100% complementary to the probe can be identified. For detailed instructions on nucleic acid hybridization, please refer to the relevant literature.
  • the stringent conditions are usually chosen to be lower than that of specific sequences.
  • the thermal melting point (T m ) at ionic strength pH is about 5-10°C.
  • T m is the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence in an equilibrium state (under the specified ionic strength, pH and nucleic acid at a concentration) (because the target sequence present in excess, at T m 50% of the probes are occupied at equilibrium) less stringent conditions may be conditions: wherein the salt concentration is 7.0 to 8.3 pH of less than about 1.0M Na The ion concentration is usually about 0.01 to 1.0M sodium ion concentration (or other salt), and the temperature is at least about 30°C for short probes (including but not limited to 10 to 50 nucleotides), and for Long probes (including but not limited to more than 50 nucleotides) are at least about 60°C.
  • Stringent conditions can also be achieved by adding destabilizing agents such as formamide.
  • the positive signal can be at least twice the background hybridization, and optionally 10 times the background hybridization.
  • Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 ⁇ SSC and 1% SDS, cultured at 42°C; or 5 ⁇ SSC, 1% SDS, cultured at 65° C., washed in 0.2 ⁇ SSC and washed in 0.1% SDS at 65° C. The washing can be performed for 5, 15, 30, 60, 120 minutes or more.
  • the “multiple” in the present invention usually means 2-8, preferably 2-4; the "replacement” refers to the replacement of one or more amino acid residues with different amino acid residues; The “deletion” refers to the decrease in the number of amino acid residues, that is, the lack of one or more amino acid residues; the “insertion” refers to the change in the sequence of amino acid residues, relative to natural molecules, The change results in the addition of one or more amino acid residues.
  • recombinant host cell strain or "host cell” means a cell containing a polynucleotide of the present invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, f-pairing, or in the art Other known methods.
  • the exogenous polynucleotide can be maintained as a non-integrating vector such as a plasmid or can be integrated into the host genome.
  • the host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.
  • operably connected refers to a functional connection between two or more elements, and operably connected elements may be contiguous or non-contiguous.
  • transformation refers to the genetic transformation of polynucleotides or polypeptides into plants in such a way as to introduce coding genes into plant cells.
  • Methods for introducing the polynucleotide or polypeptide into plants are well-known in the art, including but not limited to stable transformation methods, transient transformation methods, virus-mediated methods, and the like.
  • Stable transformation means that the introduced polynucleotide construct is integrated into the genome of the plant cell and can be inherited through its progeny;
  • transient transformation means that the polynucleotide is introduced into a plant but can only be temporarily expressed in the plant Or exist.
  • coding sequence a nucleic acid sequence transcribed into RNA.
  • recombinant plant expression vector one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art.
  • Binary vectors and vectors with helper plasmids are mostly commonly used for Agrobacterium-mediated transformation.
  • Binary vectors usually include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, and the like.
  • Figure 1 is a schematic diagram of the ZmSBP12 gene overexpression vector Ubi::ZmSBP12-eGFP.
  • Figure 2 The results of qPCR detection of ZmSBP12 gene on homozygous transgenic plants.
  • Figure 3 shows the performance of wild-type and mutant ZmSBP12-OE after drought treatment.
  • Figure 4 shows the morphological characteristics of the homozygous mutant ZmSBP12-OE.
  • the mutant ZmSBP12-OE compares with the wild-type under natural sunlight conditions: the plant height is slightly lower and the leaf spacing is reduced at the unfolding leaf stage of 1, 8; the wild-type SAM has reached the floret differentiation stage, while the mutant has just reached the spikelet differentiation. period.
  • Figure 5 shows the plant height statistics of mutant ZmSBP12-OE and wild type under natural light conditions. From the statistical data, the plant height of the mutant ZmSBP12-OE was significantly lower than that of the wild type.
  • YW784-785 represents OE-1
  • YW786-788 represents OE-2
  • YW789-790 represents OE-3
  • YW791-792 represents OE-3. From the sample size N ⁇ 30, P value ⁇ 0.005 is regarded as reaching the extremely significant level.
  • Figure 6 shows the statistics of the length of each above-ground node of mutant ZmSBP12-OE and wild-type under natural light conditions.
  • Figure 7 shows the change in the length of each aboveground node of the mutant ZmSBP12-OE relative to the length of the wild-type aboveground node under natural light conditions (it can be seen that the reduction of the aboveground nodes 1 to 3 is the most significant.
  • the data shows this phenotype Closely related to lodging resistance).
  • Figure 8 shows the statistics of plant height and ear height of F1 in combination with wild-type and ZmSBP12-OE and backbone parents (WL1, WL2, WL3, WL4, WL5). Ear height).
  • the maize inbred line "Xiang249" in the examples can be obtained by the public from the Institute of Biotechnology, Chinese Academy of Agricultural Sciences.
  • the modification method is as follows: use primers
  • 6827-F1/R1 GTTTGGTGTTACTTCTGCAGATGGAGTGGACGGCCCCGAA/GCTGAGCAGGCTCAGGGCATGCTGAAGATCCGCTGCTC; use the cDNA obtained by the above reverse transcription as a template, perform the first round of PCR amplification to obtain fragment 1; use the primer 6827-F2/R2: GCCCTGAGCCTGCTCAGCGCGCCGCTGGTTGAAA to obtain the above-mentioned TAGGCCGCCATGGTTGTGCAGCCTTTGTTGCAGT cDNA is used as a template, and the second round of PCR amplification is performed to obtain fragment 2;
  • 6827-F1/R2 GTTTGGTGTTACTTCTGCAGATGGAGTGGACGGCCCCGAA/TGCCACCACCGGATCCATTTATCTGGTTTACACCAAAGAAA;
  • the third round of PCR-overlap PCR was performed using the mixture of fragment 1 and fragment 2 as a template to obtain the gene coding sequence (SEQ ID No. 6) of ZmSPL12 after site modification.
  • the DNA molecule shown in SEQ ID No. 2 in the sequence list was inserted between the PstI and BamHI restriction sites of the modified pCAMBIA vector to obtain the ZmSBP12 overexpression vector pCAMBIA-Ubi::ZmSBP12- eGFP. And sequence the vector.
  • the sequencing results showed that the vector pCAMBIA-Ubi::ZmSBP12-eGFP inserted the DNA molecule shown in SEQ ID No. 2 in the sequence table between the PstI and BamHI restriction sites of the modified pCAMBIA, and kept the modified pCAMBIA The other sequence of the vector is not changed to the vector obtained.
  • the ZmSBP12 overexpression vector pCAMBIA-Ubi::ZmSBP12-eGFP obtained in step 1 was transferred into Agrobacterium EHA105 by electric shock in the ultraclean table to obtain the recombinant strain pCAMBIA-Ubi::ZmSBP12-eGFP/EHA105, which was used to transform Xiang249 Wild type immature embryo callus.
  • the genetic transformation method using Agrobacterium to infect maize immature embryos (1) Prepare Agrobacterium infection solution; (2) Infection and co-cultivation: Use the infection solution to infect maize immature embryos. After the infection is over, Place the immature embryo with the shield side up and place it in a co-culture medium for dark culture; (3) Screening, subculture and plant regeneration; (4) Induction; (5) Differentiation; (6) Rooting; (7) Seedling refinement, Transplanted in the field to obtain T 0 generation transgenic maize.
  • the mutant Ubi::ZmSBP12-eGFP was obtained with the DNA molecule shown in SEQ ID No. 6 in the sequence list.
  • V/V Basta solution Screening and identification by spraying 1/1000 (V/V) Basta solution, if the plant is a negative plant (not containing genetically modified ingredients), the leaves will wither after applying Basta solution for three days, while the leaves of positive plants (containing genetically modified ingredients) will not Variety.
  • the mutant ZmSBP12-OE and the wild-type were grown in a light incubator (14h light, 10h dark; temperature 28°C under light, temperature 22°C under dark) to two leaves in one heart, and began the drought treatment until the wild-type leaves all wilt and wither. After rewatering and resuming culture for 2 days, the survival rate was calculated. The results showed that the mutant ZmSBP12-OE was significantly more drought-tolerant than the wild type (Figure 3).
  • the height of the mutant ZmSBP12-OE was slightly lower than that of the wild-type plant at the eight-leaf period, and the leaf spacing was reduced (Figure 4); the growth of the plant was slow, and the wild-type SAM had reached the floret differentiation stage, while the mutant was Just reached the stage of spikelet differentiation ( Figure 4). The height of mature plants decreases. Under the same growth conditions, the length of the aboveground nodes of the mutant ZmSBP12-OE and the wild-type were calculated, and it was found that most of the internodes of the mutant were shorter than those of the wild-type, but there were also some nodes that were longer than the wild-type. Slightly longer.
  • the planting density is 6000 plants/mu, and the management is the same as general corn field management.
  • the phenotype is measured, and the statistical results are shown in Figure 8.
  • the results show that overexpression of ZmSBP12 gene can reduce the plant height and ear height of hybrids. The results indicate that the increase of ZmSBP12 gene expression can help improve the lodging density resistance.

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Abstract

提供了ZmSBP12基因在调控玉米抗旱性、株高及穗位高中的用途。将ZmSBP12基因在玉米中过表达后，玉米突变体植株出现抗旱性增加、株高及穗位高降低等性状。ZmSBP12过表达导致抗旱性增加、株高及穗位高降低，表明ZmSBP12基因对玉米的抗旱、株型(株高)等起着至关重要的作用。通过提高ZmSBP12基因的表达丰度实现提高玉米抗旱性、降低玉米株高和穗位高等效果，能应用于辅助选育抗旱及抗倒伏的玉米新品种，还可应用于玉米优良自交系和杂交种的培育。

Description

ZmSBP12基因在调控玉米抗旱性、株高及穗位高中的用途 技术领域
本发明涉及ZmSBP12基因的新用途，尤其涉及ZmSBP12基因在调控玉米抗旱性、株高以及穗位高等方面的新用途，属于ZmSBP12基因在玉米育种应用领域。
背景技术
玉米已成为中国第一大粮食作物，年种植面积达5亿亩。中国作为一个农业大国，面临着用世界7％的土地养活世界22％的人口的难题，并且近年来中国对国外进口玉米的依赖不断加重，造成严重的粮食安全隐患。因此迫切需要培育抗逆性强(抗旱)、抗倒伏的具有高产潜力的杂交种。
干旱是影响中国及世界玉米稳产的重要环境因子，世界上干旱及半干旱地区约占地球陆地面积的34.9％，占耕地面积的42.9％，每年因干旱造成粮食减产量约占各种自然灾害造成粮食损失的60％。干旱是影响中国及世界玉米稳产的重要环境因子，中国玉米近70％的面积分布在东北、华北、西南和西北依靠自然降雨的丘陵旱地或平原旱地上，种植面积最大的吉林省一般干旱年份减产25％以上，大旱年减产30％～35％，严重旱灾年部分地区几乎绝收。因此，筛选营养和水分高效利用基因型，培育水、肥资源节约型和环保型的新品种已成为中国玉米生产的迫切要求和抗旱育种的重要目标。并且随着全球变暖及环境恶化，还将有更多的地方受到干旱的威胁。为此需要加快对玉米及其他作物抗旱性的研究。
目前关于玉米抗旱方面的研究尚不完善，目前具有应用潜力的基因仅有ZmVPP1等为数不多的几个基因。长期的玉米生产实践表明，培育矮秆、耐密、适宜机械化作业的玉米新品种是增产、增效的另一关键技术措施。已有报道显示，株高升高会使更多的同化物用于营养生长而不是生殖生长，直接影响玉米产量；较高的株高还会使玉米的重心升高，并伴随植株 徒长、茎秆变细、维管束减少、细胞壁木质素纤维素的组成和含量改变、茎秆机械强度降低，进而加重倒伏的发生。因此，有效降低株高是培育高产稳产玉米新品种的基本要求。在水稻和小麦中，半矮杆基因sd1和rht1的利用直接导致了第一次绿色革命，作物的产量得到了很大的提升，然而在玉米中类似这种绿色革命基因的、既能有效降低株高又没有明显穗部负向效应的主效半矮杆基因依然没有找到。
ZmSBP12基因是一个受miR156调控的玉米SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE(SPL)转录因子。ZmSPL12基因具有4个外显子，3个内含子。其主要的功能区域包含miR156调控位点(CATGCTCTCTCTCTTCTGTCA)及SBP结构域。
玉米的抗旱性、株高、穗位高往往受到许多微效数量性状位点(QTL)的控制，现在的玉米育种依然需要通过不断累积这些微效QTL来提高抗旱性、降低株高、穗位高，选择效率低下。因此挖掘控制玉米抗旱、株高、穗位高的主效基因，解析其遗传调控网络具有重要的理论和实践意义。
发明内容
本发明的主要目的是提供ZmSBP12基因在调控玉米抗旱性、株高或穗位高中的用途；
本发明的上述目的是通过以下技术方案来实现的：
本发明将ZmSBP12基因在玉米中过表达后，玉米突变体植株出现抗旱性增加、株高及穗位高降低等性状。ZmSBP12基因过表达导致抗旱性增加、株高及穗位高降低，表明ZmSBP12基因对玉米的抗旱、株型(株高)等起着至关重要的作用。
因此，通过改变ZmSBP12基因在玉米中的表达丰度能够调控玉米的抗旱性增加、株高及穗位高；譬如，通过提高ZmSBP12在玉米中的表达丰度能够提高玉米抗旱性、降低玉米株高或穗位高；相反，降低ZmSBP12 基因在玉米中的表达丰度，则能够降低玉米抗旱性、使玉米株高或穗位高升高。
本发明通过提高ZmSBP12基因的表达丰度达到了提高玉米抗旱性、降低玉米株高和穗位高的效果，因此，所有涉及通过调控ZmSBP12基因的mRNA和蛋白丰度来提高玉米抗旱性、降低玉米株高和穗位高的方法都应属于本发明的保护范围，这里所述的方法应该包括所有人工合成和设计核苷酸或蛋白的方法及利用未曾报道的自然变异。
至于如何提高ZmSBP12基因在玉米中的表达丰度以及降低ZmSBP12基因在玉米中的表达丰度，这均是本领域技术人员可以通过各种常规的技术手段来实现，譬如，通过构建ZmSBP12基因的过表达载体，使ZmSBP12基因在玉米中过表达或超表达，进而使玉米的抗旱性提高或降低玉米的株高或穗位高，进而选育得到抗逆性强(抗旱)、抗倒伏的转基因植物；或者通过基因编辑技术或基因敲除技术等将植物体内的ZmSBP12基因进行功能缺陷突变得到不抗旱、株高及穗位高升高的转基因植物。
本发明还涉及所述ZmSBP12基因在玉米育种中的应用；包括玉米自交系和杂交种的培育过程中；具体的，在玉米杂交种其中一个亲本中过表达ZmSBP12基因即可达到降低玉米株高、穗位高，提高玉米抗旱性的效果。
本发明提供了一种提高玉米抗旱的方法，包括：(1)构建含有所述ZmSBP12基因的重组植物表达载体；(2)将所构建的重组植物表达载体转化到植物组织或植物细胞中；(3)将ZmSBP12基因在植物组织或细胞中进行过表达。
本发明还提供了提高玉米抗倒伏的方法，包括：(1)构建含有所述ZmSBP12基因的重组植物表达载体；(2)将所构建的重组植物表达载体转化到植物组织或植物细胞中；(3)将ZmSBP12基因在植物组织或细胞中进行过表达。
本发明中所述提高玉米抗旱性、降低株高及穗位高体现在转基因植 物与野生型在相同条件下抗旱性增强、株高及穗位高降低。
将所述ZmSBP12基因可操作的与表达调控元件相连接，得到可以在植物中表达该编码基因的重组植物表达载体；该重组植物表达载体可以由5′端非编码区、SEQ ID No.2所示的多核苷酸序列和3′非编码区组成，其中，所述的5′端非编码区可以包括启动子序列、增强子序列或/和翻译增强序列；所述的启动子可以是组成性启动子、诱导型启动子、组织或器官特异性启动子；所述的3′非编码区可以包含终止子序列、mRNA切割序列等。合适的终止子序列可取自根癌农杆菌的Ti-质粒，例如章鱼碱合成酶和胭脂碱合成酶终止区。
所述重组植物表达载体还可含有用于选择转化细胞的选择性标记基因。选择性标记基因用于选择经转化的细胞或组织。标记基因包括：编码抗生素抗性的基因以及赋予除草化合物抗性的基因等。此外，所述的标记基因还包括表型标记，例如β-半乳糖苷酶和荧光蛋白等。
另外，本领域技术人员可以将SEQ ID No.2所示的多核苷酸进行优化以增强在植物中的表达效率。例如，可采用目标植物的偏爱密码子进行优化来合成多核苷酸以增强在目标植物中的表达效率；或者将SEQ ID No.2所示的多核苷酸进行位点改造得到改造后的变体，作为一种优选的实施方案，所述变体的多核苷酸序列为SEQ ID No.6所示。
本发明还提供了含有ZmSBP12基因的重组植物表达载体以及含有该重组植物表达载体的宿主细胞。
本发明中所述ZmSBP12基因的编码氨基酸序列为SEQ ID No.1所示。由于氨基酸序列的特殊性，任何含有SEQ ID NO.1所示氨基酸序列的肽蛋白的片段或其变体，如其保守性变体、生物活性片段或衍生物，只要该肽蛋白的片段或肽蛋白变体与前述氨基酸序列同源性在90％以上，均属于本发明保护范围之列。具体的所述改变可包括氨基酸序列中氨基酸的缺失、插入或替换；其中，对于变体的保守性改变，所替换的氨基酸具有 与原氨基酸相似的结构或化学性质，如用亮氨酸替换异亮氨酸，变体也可具有非保守性改变，如用色氨酸替换甘氨酸。
所述ZmSBP12基因的核苷酸序列为SEQ ID No.2所示。由于核苷酸序列的特殊性，任何SEQ ID NO.2所示多核苷酸的变体，只要其与该多核苷酸具有90％以上同源性，均属于本发明保护范围之列。所述多核苷酸的变体是指一种具有一个或多个核苷酸改变的多核苷酸序列。此多核苷酸的变体可以使生的变位变异体或非生的变异体，包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个多核苷酸的取代、缺失或插入，但不会从实质上改变其编码的肽蛋白的功能。
本发明还涉及检测ZmSBP12基因表达量的特异性扩增引物，其序列分别为SEQ ID No.3和SEQ ID No.4所示；
上游引物：5’-AGCTCATCTGACTTAAAGCCCC-3’
下游引物：5’-TTCATTGGCCAAGGCTCATCT-3’
利用该特异性扩增引物对玉米进行扩增，其产物片段为155bp，产物序列为SEQ ID No.5所示。
本发明通过提高ZmSBP12基因的表达丰度达到了提高玉米抗旱性、降低玉米株高和穗位高的效果，能够应用于辅助选育抗旱及抗倒伏的玉米新品种，还可应用于玉米优良自交系和杂交种的培育。
本发明所涉及到的术语定义
除非另外定义，否则本文所用的所有技术及科学术语都具有与本发明所属领域的普通技术人员通常所了解相同的含义。虽然在本发明的实践或测试中可使用与本文所述者类似或等效的任何方法、装置和材料，但现在描述优选方法、装置和材料。
术语“多核苷酸”或“核苷酸”意指单股或双股形式的脱氧核糖核苷酸、脱 氧核糖核苷、核糖核苷或核糖核苷酸及其聚合物。除非特定限制，否则所述术语涵盖含有天然核苷酸的已知类似物的核酸，所述类似物具有类似于参考核酸的结合特性并以类似于天然产生的核苷酸的方式进行代谢。除非另外特定限制，否则所述术语也意指寡核苷酸类似物，其包括PNA(肽核酸)、在反义技术中所用的DNA类似物(硫代磷酸酯、磷酰胺酸酯等等)。除非另外指定，否则特定核酸序列也隐含地涵盖其保守修饰的变异体(包括(但不限于)简并密码子取代)和互补序列以及明确指定的序列。特定而言，可通过产生其中一个或一个以上所选(或所有)密码子的第3位经混合碱基和/或脱氧肌苷残基取代的序列来实现简并密码子取代(Batzer等人,Nucleic Acid Res.19:5081(1991)；Ohtsuka等人,J.Biol.Chem.260:2605-2608(1985)；和Cassol等人,(1992)；Rossolini等人,Mol Cell.Probes 8:91-98(1994))。
术语“多肽”、“肽”和“蛋白”在本文中互换使用以意指氨基酸残基的聚合物。即，针对多肽的描述同样适用于描述肽和描述蛋白，且反之亦然。所述术语适用于天然产生氨基酸聚合物以及其中一个或一个以上氨基酸残基为非天然编码氨基酸的氨基酸聚合物。如本文中所使用，所述术语涵盖任何长度的氨基酸链，其包括全长蛋白(即抗原)，其中氨基酸残基经由共价肽键连接。
所述“变体”意指基本相似的序列，对于多核苷酸，变体包含天然多核苷酸中一个或多个位点处一个或多个核苷酸的缺失、插入或/和替换。对于多核苷酸，保守的变体包括由于遗传密码的简并性而不改变编码的氨基酸序列的那些变体。诸如此类天然存在的变体可通过现有的分子生物学技术来鉴定。变体多核苷酸还包括合成来源的多核苷酸，例如采用定点诱变所得到的仍编码SEQ ID No.1所示氨基酸序列的多核苷酸变体或者是通过重组的方法(例如DNA改组)。本领域技术人员可通过以下分子生物技术手段来筛选或评价变体多核苷酸所编码蛋白的功能或活性：DNA结合活性、蛋白之间的相互作用，瞬时研究中基因表达的激活情况或转基因植物中表达的效应等。
本发明中所述“严谨杂交条件”意指在所属领域中已知的低离子强度和高温的条件。通常，在严谨条件下，探针与其靶序列杂交的可检测程度比与其它序列杂交的可检测程度更高(例如超过本底至少2倍。严谨杂交条件是序列依赖性的，在不同的环境条件下将会不同，较长的序列在较高温度下特异性杂交。通过控制杂交的严谨性或洗涤条件可鉴定与探针100％互补的靶序列。对于核酸杂交的详尽指导可参考有关文献(Tijssen,Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes,"Overview of principles of hybridization and the strategy of nucleic acid assays.1993)。更具体的，所述严谨条件通常被选择为低于特异序列在规定离子强度pH下的热熔点(T m)约5-10℃。T m为在平衡状态下50％与目标互补的探针杂交到目标序列时所处的温度(在指定离子强度、pH和核酸浓度下)(因为目标序列过量存在，所以在T m下在平衡状态下50％的探针被占据)。严谨条件可为以下条件：其中在pH 7.0到8.3下盐浓度低于约1.0M钠离子浓度，通常为约0.01到1.0M钠离子浓度(或其它盐)，并且温度对于短探针(包括(但不限于)10到50个核苷酸)而言为至少约30℃，而对于长探针(包括(但不限于)大于50个核苷酸)而言为至少约60℃。严谨条件也可通过加入诸如甲酰胺的去稳定剂来实现。对于选择性或特异性杂交而言，正信号可为至少两倍的背景杂交，视情况为10倍背景杂交。例示性严谨杂交条件可如下：50％甲酰胺，5×SSC和1％SDS，在42℃下培养；或5×SSC，1％SDS，在65℃下培养，在0.2×SSC中洗涤和在65℃下于0.1％SDS中洗涤。所述洗涤可进行5、15、30、60、120分钟或更长时间。
本发明中所述的“多个”通常意味着2-8个，优选为2-4个；所述的“替换”是指分别用不同的氨基酸残基取代一个或多个氨基酸残基；所述的“缺失”是指氨基酸残基数量的减少，也即是分别缺少其中的一个或多个氨基酸残基；所述的“插入”是指氨基酸残基序列的改变，相对天然分子而言，所述改变导致添加一个或多个氨基酸残基。
术语“重组宿主细胞株”或“宿主细胞”意指包含本发明多核苷酸的细胞，而不管使用何种方法进行插入以产生重组宿主细胞，例如直接摄取、转导、f配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。宿主细胞可为原核细胞或真核细胞，宿主细胞还可为单子叶或双子叶植物细胞。
术语“可操作的连接”指两个或更多个元件之间功能性的连接，可操作的连接的元件可为邻接或非邻接的。
术语“转化”指将编码基因导入到植物细胞内部这样的方式将多核苷酸或多肽遗传转化到植物中。将所述多核苷酸或多肽引入到植物中的方法为本领域所习知，包括但不限于稳定转化法、瞬时转化法和病毒介导法等。“稳定转化”指被引入的多核苷酸构建体整合至植物细胞的基因组中并能通过其子代遗传；“瞬时转化”指多核苷酸被引入到植物中但只能在植物中暂时性表达或存在。
术语“表达”：内源性基因或转基因在植物细胞中的转录和/或翻译。
术语“编码序列”：转录成RNA的核酸序列。
术语“重组植物表达载体”：一种或多种用于实现植物转化的DNA载体；本领域中这些载体常被称为二元载体。二元载体连同具有辅助质粒的载体是大多常用于土壤杆菌介导转化的。二元载体通常包括：T-DNA转移所需要的顺式作用序列、经工程化处理以便能够在植物细胞中表达的选择标记物，待转录的异源性DNA序列等。
附图说明
图1为ZmSBP12基因过表达载体Ubi::ZmSBP12-eGFP的示意图。
图2对纯合体转基因植株进行ZmSBP12基因的qPCR检测结果。
图3为野生型及突变体ZmSBP12-OE经干旱处理后的表现。
图4为纯合突变体ZmSBP12-OE的形态学特征。突变体ZmSBP12-OE在自然日照条件下与野生型比较：1、8展开叶时期，植株高度略低，叶间距减小；野生型SAM已达到小花分化时期，而突变体则刚达到小穗分化时期。
图5为自然光照条件下，突变体ZmSBP12-OE与野生型的株高统计。从统计数据看，突变体ZmSBP12-OE的株高显著低于野生型。图中YW784-785代表OE-1；YW786-788代表OE-2；YW789-790代表OE-3；YW791-792代表OE-3。从样本量N≥30，P值≤0.005视为达到极显著水平。
图6为自然光照条件下，突变体ZmSBP12-OE与野生型的地上各节长度的统计。
图7为自然光照条件下，突变体ZmSBP12-OE的地上各节长度相对于野生型的地上各节长度的变化量(可以看出地上1～3节降低最为显著，该数据显示这一表型与抗倒伏密切相关)。
图8为野生型及ZmSBP12-OE与骨干亲本(WL1、WL2、WL3、WL4、WL5)组配F1的株高、穗位高统计(可以看ZmSBP12-OE也可以显著降低杂交种的株高和穗位高)。
具体实施方式
以下结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例中所使用的实验方法如无特殊说明，均为常规方法。
实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例中的玉米自交系“Xiang249”公众可以从中国农业科学院生物技术研究所获得。
实施例1 ZmSBP12基因的获得及突变体ZmSBP12-OE的构建
一.ZmSBP12基因的获得及改造
利用Trizol法(参考赛默飞世尔试剂公司Trizol说明书)提取B73自交系V2时期植株的总RNA，反转录(参考天根生化科技反转录试剂盒(KR106-02)说明书)。由于ZmSBP12基因是一个受miR156调控的玉米SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE(SPL)转录因子，所以需要见miR156调控位点(CATGCTCTCTCTCTTCTGTCA)进行改造，以使miR156不能识别切割ZmSBP12。
改造方法如下：利用引物
6827-F1/R1：GTTTGGTGTTACTTCTGCAGATGGAGTGGACGGCCCCGAA/GCTGAGCAGGCTCAGGGCATGCTGAAGATCCGCTGCTC；以上述反转录得到的cDNA为模板，进行第一轮PCR扩增得到片段1；利用引物6827-F2/R2:GCCCTGAGCCTGCTCAGCGCCGGCGCTTGTGGACTGCCTGATT/TGCCACCACCGGATCCATTTATCTGGTTTACACCAAAGAAA以上述反转录得到的cDNA为模板，进行第二轮PCR扩增得到片段2；
再利用引物
6827-F1/R2：GTTTGGTGTTACTTCTGCAGATGGAGTGGACGGCCCCGAA/TGCCACCACCGGATCCATTTATCTGGTTTACACCAAAGAAA；
以上述片段1和片段2的混合物为模板进行第三轮PCR-重叠PCR得到位点改造后的ZmSPL12的基因编码序列(SEQ ID No.6)。
二突变体ZmSBP12-OE的创制及生物学特征
1、ZmSBP12过表达载体pCAMBIA-Ubi::ZmSBP12-eGFP的构建
采用同源重组的方法将序列表中SEQ ID No.2所示的DNA分子插入 到改造后的pCAMBIA载体的PstI和BamHI酶切位点间，获得了ZmSBP12过表达载体pCAMBIA-Ubi::ZmSBP12-eGFP。并对载体进行测序。测序结果表明：载体pCAMBIA-Ubi::ZmSBP12-eGFP为将序列表中SEQ ID No.2所示的DNA分子插入到改造后的pCAMBIA的PstI和BamHI酶切位点间，且保持改造后的pCAMBIA载体的其他序列不发生改变得到的载体。
2、重组菌的获得
将步骤1获得的ZmSBP12过表达载体pCAMBIA-Ubi::ZmSBP12-eGFP在超净台内通过电击的方法转入农杆菌EHA105中得到重组菌pCAMBIA-Ubi::ZmSBP12-eGFP/EHA105，用于转化Xiang249野生型幼胚愈伤。
3、突变体ZmSBP12-OE的构建
利用农杆菌侵染玉米幼胚的遗传转化方法：(1)制备农杆菌侵染液；(2)侵染与共培养：用所述侵染液对玉米幼胚进行侵染，侵染结束后，将幼胚盾面朝上，放入共培养培养基中暗培养；(3)筛选、继代和植株再生；(4)诱导；(5)分化；(6)生根；(7)炼苗，转田间种植，得到T 0代转基因玉米。
参照上述方法，以序列表中SEQ ID No.6所示的DNA分子获得突变体Ubi::ZmSBP12-eGFP。
4、转基因突变体ZmSBP12-OE的鉴定
通过喷洒1/1000(V/V)的Basta溶液进行筛选鉴定，若植株为阴性植株(不含有转基因成分)则叶片在涂抹Basta溶液三天后会枯萎，而阳性植株(含有转基因成分)的叶片无变化。
5、突变体ZmSBP12-OE的抗旱性鉴定
将突变体ZmSBP12-OE与野生型在光照培养箱(14h光照，10h黑暗；光照下温度28℃，黑暗下温度22℃)生长至两叶一心，开始干旱处理，至野生型叶片全部萎蔫干枯，复水，恢复培养2天，统计成活率，结果表 明突变体ZmSBP12-OE比野生型显著耐旱(图3)。
6、突变体ZmSBP12-OE的形态学特征
在自然日照条件下，8展开叶时期，突变体ZmSBP12-OE较野生型植株高度略低，叶间距减小(图4)；植株发育迟缓，野生型SAM已达到小花分化时期，而突变体则刚达到小穗分化时期(图4)。成熟植株株高降低。在相同生长条件下，对突变体ZmSBP12-OE与野生型的地上各节长度的统计，发现突变体大部分节间长度较野生型都有所下降，但也有一些节的节间长度较野生型略长。同时，对突变体ZmSBP12-OE的地上各节长度相对于野生型的地上各节长度的变化量做了统计，发现突变体的地上1～3节降低最为显著，生产实践及研究表明，玉米的茎折即发生在地上1～3节并且茎折会造成玉米减产甚至绝产，这一表型与抗倒伏密切相关。
7、过表达ZmSBP12可降低杂交种的株高和穗位高
通过将野生型及ZmSBP12-OE突变体与骨干亲本(WL1、WL2、WL3、WL4、WL5)组配F1，并于廊坊进行表型观察，种植密度6000株/亩，管理同一般玉米大田管理，待株形固定后(授粉后30天)进行表型测量，统计结果如图8。结果表明，过表达ZmSBP12基因可降低杂交种的株高和穗位高，该结果表明ZmSBP12基因的表达量的提高有助于提高抗倒伏密性能。
综上所述，将ZmSBP12基因在玉米中进行过表达后，玉米突变体植株的抗旱性增加、株高及穗位高降低，表明ZmSBP12基因对玉米的抗旱、株型(株高)等起着至关重要的作用且具有重大的育种应用价值。

Claims (10)

  1. ZmSBP12基因或其变体在调控玉米抗旱性中的用途。
  2. ZmSBP12基因或其变体在调控株高或穗位高中的用途。
  3. 按照权利要求1所述的用途，其特征在于，所述的调控玉米抗旱性是提高玉米的抗旱性或降低玉米的抗旱性。
  4. 按照权利要求2所述的用途，其特征在于，所述的调控株高或穗位高是降低玉米株高或穗位高，或者是使玉米株高或穗位高提升。
  5. 一种提高植物抗旱性的方法，包括：(1)构建含有ZmSBP12基因的重组植物表达载体；(2)将所构建的重组植物表达载体转化到植物组织或植物细胞中；(3)将ZmSBP12基因在植物组织或细胞中进行过表达；优选的，所述植物是玉米。
  6. 一种提高植物抗倒伏的方法，包括：(1)构建含有ZmSBP12基因的重组植物表达载体；(2)将所构建的重组植物表达载体转化到植物组织或植物细胞中；(3)将ZmSBP12基因在植物组织或细胞中进行过表达；优选的，所述植物是玉米。
  7. 一种降低植物抗旱性的方法，其特征在于，包括：通过基因编辑技术或基因敲除技术将玉米中的ZmSBP12基因进行功能缺陷突变；优选的，所述植物是玉米。
  8. 一种使玉米株高或穗位高提升的方法，其特征在于，包括：通过基因编辑技术或基因敲除技术将玉米中的ZmSBP12基因进行功能缺陷突变。
  9. 按照权利要求1或2所述的用途，其特征在于，所述ZmSBP12基因的核苷酸序列是SEQ ID No.2所示，其编码的蛋白的氨基酸序列为SEQ ID No.1所示；所述ZmSBP12基因的变体的核苷酸序列是SEQ ID No.6所示。
  10. 检测ZmSBP12基因表达量的特异性扩增引物，其特征在于，其核苷酸序列分别为SEQ ID No.3和SEQ ID No.4所示。
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CN115160424A (zh) * 2022-05-10 2022-10-11 华中农业大学 Zm00001d022481基因在玉米株高发育中的应用
CN115160424B (zh) * 2022-05-10 2024-05-24 华中农业大学 Zm00001d022481基因在玉米株高发育中的应用
CN114736280A (zh) * 2022-05-24 2022-07-12 中国农业大学 ZmROA1蛋白在调控植物耐密性中的应用
CN114736280B (zh) * 2022-05-24 2023-03-24 中国农业大学 ZmROA1蛋白在调控植物耐密性中的应用
CN117447575A (zh) * 2023-12-19 2024-01-26 中国农业大学 深根蛋白在特异性调控玉米根夹角中的应用
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