Application of grape nitrate transporter NRT1.1 and cyclic nucleotide-gated ion channel CNGC15 in improving nitrogen utilization

Abstract

The application discloses application of VvNRT1.1 and VvCNGC15 proteins in improving nitrogen utilization rate of grape, and belongs to the technical field of plant genetic engineering and molecular breeding. The application proves that the VvNRT1.1 and VvCNGC15 proteins directly physically interact, and overexpression of the VvNRT1.1 or VvCNGC15 can improve the absorption and assimilation efficiency of nitrate nitrogen of grape. Therefore, the VvNRT1.1 and VvCNGC15 can be used as key molecular targets for improving nitrogen utilization rate, and have potential application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically involving the application of grape nitrate transporter NRT1.1 and cyclic nucleotide-gated ion channel CNGC15 in improving nitrogen use efficiency, and particularly involving a method for improving grape nitrogen use efficiency by utilizing the protein interaction between VvNRT1.1 and VvCNGC15 and its application in grape genetic improvement. Background Technology

[0002] The information disclosed in this background section is intended only to enhance some understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art.

[0003] Grape( Vitis vinifera Grapes (Vitis vinifera) belong to the genus Vitis in the family Vitaceae and are one of the most economically viable fruit tree species in my country. In 2022, my country's grape planting area was approximately 705,100 hectares, with a yield of about 15.3779 million tons, ranking first in the world in grape production. Excessive application of nitrogen fertilizer not only reduces nitrogen nutrient responsiveness and utilization rate, increasing grape cultivation costs, but also leads to low soil organic matter content, soil acidification, and mineral nutrient imbalance, threatening the healthy and sustainable development of the grape industry. Therefore, improving the responsiveness of fruit trees to nitrogen fertilizer supply, breeding nitrogen-efficient grape varieties, and achieving efficient utilization of chemical fertilizer nutrients are important goals of fruit tree genetic improvement.

[0004] Nitrate nitrogen (NO3) – Nitrate is the primary nitrogen source for grapes and an important signaling molecule. The nitrate transporter NRT1.1 (also known as NPF6.3) acts as a NO3-transfer protein. – Its "transceivers" (transporter proteins and receptor proteins) can transport and sense NO3. – This, in turn, triggers numerous physiological changes in response to external NO3. – Concentration changes reflect the absorption and distribution of NO3 by plants. – The key steps, thereby improving NO3 – This lays the foundation for the effective utilization rate.

[0005] Recent studies in the model plant Arabidopsis thaliana have found that NRT1.1 plays a role in sensing NO3. – It can then activate some unknown plasma membrane Ca 2+ Channel, and produce NO3 – Induced Ca 2+ Characteristic current (Wang et al., 2021). However, the Ca described in this study... 2+The molecular identity of the channel has not yet been identified, and there are no publicly available reports on whether NRT1.1 has direct protein-protein interactions with any CNGC family members.

[0006] Calcium ions (Ca 2+ Cyclic nucleotide-gated ion channels (CNGCs) are ubiquitous second messengers in plant cells, widely involved in growth, development, and stress signal transduction. They are a class of channels that regulate Ca2+ levels. 2+ Non-selective cation channels with permeability have been reported to participate in processes such as root hair development, pollen tube growth, and immune response in Arabidopsis thaliana.

[0007] However, to date, no CNGC family member has been reported to participate in nitrate signal transduction, and no literature reveals a direct protein-protein interaction between NRT1.1 and CNGCs. As a perennial woody fruit tree, grapes exhibit fundamentally different mechanisms of nitrogen absorption, storage, and reuse compared to annual herbaceous plants such as Arabidopsis thaliana. The specific biological functions of NRT1.1 in grapes, and whether it participates in calcium... 2+ The molecular identity of signal regulation and the calcium channel proteins that interact with it is completely unknown internationally.

[0008] Through long-term and in-depth research, the inventors cloned the VvNRT1.1 and VvCNGC15 genes from grapes and confirmed that they interact directly with each other on the cell membrane. Based on this, the inventors further demonstrated that overexpression of either VvNRT1.1 or VvCNGC15 significantly enhanced the grape root system's resistance to NO3-. – and Ca 2+ This process upregulates the absorption of nitrate reductase, upregulates the expression of the nitrate reductase gene NIA1 and NR enzyme activity, ultimately significantly improving the absorption of grapes. 15 NO3 – Utilization rate.

[0009] Therefore, the technical problem to be solved by the present invention is to provide key gene targets VvNRT1.1 and VvCNGC15 in grapes to improve nitrogen utilization through protein interaction, and to provide a method for their application in grape genetic improvement. Summary of the Invention

[0010] In view of this, the present invention provides the application of the nitrate transporter VvNRT1.1 and the cyclic nucleotide-gated ion channel VvCNGC15 in improving nitrogen utilization in grapes. Overexpression vectors pBWA(V)kS-35S::VvNRT1.1-GFP and pBWA(V)kS-35S::VvCNGC15-GFP were constructed and transformed into grapes (Thompson Seedless) via Agrobacterium-mediated genetic transformation. Ion flow rate measurements revealed that the transgenic lines overexpressing VvNRT1.1 had significantly higher NO3- content.– The increased absorption rate indicates that VvNRT1.1 is effective in NO3 absorption. – Absorption function; transgenic lines overexpressing VvNRT1.1 and VvCNGC15, their Ca 2+ The absorption rates were higher than those of the wild type, indicating that overexpression of VvNRT1.1 or VvCNGC15 could promote the absorption of Ca by grape roots. 2+ Absorption.

[0011] Building upon this, the present invention further confirmed, through yeast two-hybrid assays, bimolecular fluorescence complementation, and immunoprecipitation, that VvNRT1.1 and VvCNGC15 proteins directly interact physically on the cell membrane. This result indicates a direct molecular-level correlation between the nitrate signaling pathway mediated by VvNRT1.1 and the calcium ion signaling pathway mediated by VvCNGC15.

[0012] The technical solution adopted in this invention is as follows: The first aspect provides the application of nucleotides encoding the nitrate transporter VvNRT1.1 and / or nucleotides encoding the cyclic nucleotide-gated ion channel VvCNGC15 in improving the nitrogen utilization of grapes.

[0013] In one or more embodiments of the present invention, the nitrate transporter VvNRT1.1 and the cyclic nucleotide-gated ion channel VvCNGC15 interact directly physically.

[0014] In one or more embodiments of the present invention, the amino acid sequence of the grape nitrate transporter VvNRT1.1 is shown in SEQ ID No. 2; and the amino acid sequence of the grape cyclic nucleotide-gated ion channel VvCNGC15 is shown in SEQ ID No. 4.

[0015] The nucleotide sequence encoding the VvNRT1.1 protein is shown in SEQ ID No. 1; the nucleotide sequence encoding the VvCNGC15 protein is shown in SEQ ID No. 3.

[0016] Secondly, a method for improving nitrogen utilization in grapes is provided, which achieves the promoting effect of protein interaction on nitrogen utilization by enhancing the expression of VvNRT1.1 and VvCNGC15 genes or the activity of proteins in grapes.

[0017] In one or more embodiments of the present invention, the method for enhancing the expression of VvNRT1.1 and VvCNGC15 genes or the activity of proteins in grapes includes the following steps: (1) Construct a recombinant expression vector containing a nucleotide sequence encoding the VvNRT1.1 protein, and / or a recombinant expression vector containing a nucleotide sequence encoding the VvCNGC15 protein; (2) Transform grape cells, tissues or organs with the recombinant expression vector constructed in step (1); (3) Screening to obtain transgenic grape plants that overexpress the VvNRT1.1 gene and / or the VvCNGC15 gene.

[0018] Preferably, the backbone vector of the recombinant expression vector is pBWA(V)KS, and the promoter is CaMV 35S promoter.

[0019] Thirdly, the application of biological materials containing enhanced expression of VvNRT1.1 and / or VvCNGC15 genes in improving nitrogen utilization in grapes is provided; said biological materials include expression cassettes, vectors, host bacteria, nucleic acid molecules, or kits.

[0020] Compared with the related technologies known to the inventors, one of the technical solutions of the present invention has the following beneficial effects: (1) Provide key molecular targets for nitrogen-efficient breeding of grapes This invention cloned the full-length coding sequences of the VvNRT1.1 and VvCNGC15 genes from the woody fruit tree grape and confirmed that the two genes directly interact physically on the cell membrane, providing two molecular targets with a clear interaction relationship for high-nitrogen-efficiency breeding of grapes.

[0021] (2) Verify the nitrogen-efficient function of VvNRT1.1 in grapes As a perennial woody fruit tree, grapes exhibit fundamentally different mechanisms for nitrogen absorption, storage, and reuse compared to herbaceous plants. This invention, through stable genetic transformation, demonstrates that overexpression of VvNRT1.1 in grapevines significantly promotes root NO3 uptake. – The absorption of nitrate reductase upregulates the expression of the nitrate reductase gene NIA1 and the activity of NR enzyme, thereby increasing its absorption. 15 NO3 – The utilization rate was significantly higher than that of the wild-type control. This result indicates that VvNRT1.1 is a candidate gene for genetically improving nitrogen efficiency in grapes.

[0022] (3) Revealing that CNGC15 family members are involved in nitrate signal transduction This invention confirms that VvCNGC15 is affected by NO3. – Rapidly induced expression, and its overexpression significantly enhances the resistance of grape roots to NO3. – and Ca 2+ The absorption of NIA1 upregulates NIA1 expression and NR activity, thereby increasing the absorption of NIA1 and NR activity 15 NO3 – Utilization rate.

[0023] (4) It provides two alternative target sites and their interaction information for molecular breeding. This invention validated the nitrogen-efficient functions of single genes VvNRT1.1 and VvCNGC15, and confirmed that they form functionally linked protein pairs through direct physical interaction. This discovery provides molecular evidence for a deeper understanding of the coupling mechanism between nitrate and calcium signaling, and also offers important theoretical basis and candidate gene resources for molecular breeding of nitrogen-efficient grape germplasm.

[0024] (5) Provide genetic resource reserves for nitrogen-efficient grape breeding The VvNRT1.1 and VvCNGC15 gene resources and their application methods provided by this invention can be used to cultivate new nitrogen-efficient grape varieties. Attached Figure Description

[0025] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0026] Figure 1 NO3 provided for this invention – Induced CNGC15 expression map; where A: transcriptome analysis, B: NO3. – Expression analysis of the CNGC family and C:NO3 – Expression of VvCNGC15.

[0027] Figure 2 Subcellular localization maps of VvNRT1.1 and VvCNGC15 provided for this invention; wherein A: subcellular localization map of VvNRT1.1 (A) in tobacco leaves, and B: subcellular localization map of VvCNGC15 in tobacco leaves.

[0028] Figure 3 The diagram shows the interaction between VvNRT1.1 and VvCNGC15 proteins provided by this invention; where A: yeast double hybridization demonstrates the interaction between VvNRT1.1 and VvCNGC15, B: BiFC verifies the interaction between VvNRT1.1 and VvCNGC15, and C: Co-IP verifies the interaction between VvNRT1.1 and VvCNGC15.

[0029] Figure 4The images provided for the present invention are of the overexpression vectors pBWA(V)kS-35S::VvNRT1.1-GFP and pBWA(V)kS-35S::VvCNGC15-GFP, wherein A: pBWA(V)kS-35S::VvNRT1.1-GFP overexpression vector and B: pBWA(V)kS-35S::VvCNGC15-GFP overexpression vector.

[0030] Figure 5 The images provided by this invention show transgenic grapes overexpressing VvNRT1.1 and VvCNGC15, where A: phenotypic diagram of transgenic grapes overexpressing VvNRT1.1, and B: phenotypic diagram of transgenic grapes overexpressing VvCNGC15.

[0031] Figure 6 NO3 in genetically modified grapes provided by this invention – and Ca 2+ Flow direction and velocity, where NO3 in A:VvNRT1.1-OE and VvCNGC15-OE – Flow direction, flow velocity, B: VvNRT1.1-OE and VvCNGC15-OE transgenic grape Ca 2+ Flow direction and velocity.

[0032] Figure 7 The phenotypic diagram of the transgenic grape provided by this invention, wherein A: VvNRT1.1-OE transgenic grape line NIA1 B: Expression of VvNRT1.1-OE transgenic grape lines; C: NR activity of VvNRT1.1-OE transgenic grape lines. 15 NO3 – Utilization rate, D:VvCNGC15-OE transgenic grape line NIA1 E: NR activity of VvCNGC15-OE transgenic grape lines; F: NR activity of VvCNGC15-OE transgenic grape lines. 15 NO3 – Utilization rate. Detailed Implementation

[0033] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0034] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the exemplary embodiments of the present invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0035] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0036]

[0037]

[0038] Example 2: Discovery of NO3 using transcriptome analysis – In the treated grape seedlings CNGC15 Expression was significantly upregulated ( Figure 1 A) Quantitative real-time PCR analysis was performed on CNGC family members (20 members in total) in grapes. VvCNGC15 Strongly respond to NO3 − ( Figure 1 B); Further verification using quantitative real-time PCR revealed NO3. – Inducible VvCNGC15 The expression ( Figure 1 C).

[0039] Using the grape variety 'Sunshine Rose' as the experimental material, RNA was extracted using TRIzol reagent (Invitrogen, USA). The extracted RNA was then reverse transcribed using the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) reverse transcription kit (Takara, Beijing). The transcription was performed using the LightCycler 480 I system (Roche, Switzerland) and TB Green® Premix Ex Taq. TM qRT-PCR reactions were performed using the Tli RNaseH Plus kit (Takara, Beijing), with each reaction performed in triplicate. VvActin7 was used as an internal control gene. Quantitative primers (see Table 1) were designed using the online software Primer Premier 6 and synthesized by BGI Genomics Co., Ltd. (Shenzhen). Relative expression levels were calculated using a 22... –ΔΔCt Law.

[0040] Table 1 Primer sequences

[0041] qRT-PCR system: see Table 2.

[0042] Table 2 qRT-PCR reaction system

[0043] RT-PCR procedure: see Table 3.

[0044] Table 3 qRT-PCR reaction procedure

[0045] Example 3: To analyze the potential functional sites of VvNRT1.1 and VvCNGC15, subcellular localization analysis was performed using 'Sunshine Rose' ( Shine Muscat Using root cDNA as a template, the coding sequences of VvNRT1.1 and VvCNGC15 were amplified using a high-fidelity enzyme (Novozymes, 2×Phanta Max Master Mix, P515-01) (CDS, without stop codon, primers are shown in Table 1, pBWA(V)KS-NRT1.1-osgfp and pBWA(V)KS-CNGC15-osgfp, amplification system is shown in Table 4, amplification program is shown in Table 5). Subsequently, the amplified and recovered products of VvNRT1.1 and VvCNGC15 were ligated into the linearized pBWA(V)KS-35S::osgfp vector using a one-step cloning kit (Novozymes, ClonExpress II One Step Cloning Kit, C112-01), respectively, to obtain pBWA(V)KS-35S::VvNRT1.1-osgfp and pBWA(V)KS-35S::VvCNGC15-osgfp (see Appendix). Figure 4 ).

[0046] The enzyme digestion system of pBWA(V)KS-35S::osgfp vector is shown in Table 6.

[0047] The connection system of homologous recombination is shown in Table 7.

[0048] Table 4

[0049] Table 5

[0050] Table 6

[0051] Table 7

[0052] The obtained pBWA(V)KS-35s::VvNRT1.1-osgfp and pBWA(V)KS-35S::VvCNGC15-osgfp were introduced into tobacco leaves, respectively. The green fluorescence signal of the GFP-tagged protein was observed using a confocal laser scanning microscope (Nikon C2-ER). Subcellular localization was assessed using chlorophyll localization marker co-expression, with excitation and emission wavelengths of 640 nm and 675 nm, respectively.

[0053] The results showed that both VvNRT1.1 and VvCNGC15 are located on the cell membrane. (Appendix) Figure 2 ).

[0054] Example 4: (1) Y2H detection of the interaction between VvNRT1.1 and VvCNGC15 proteins Construct bait plasmids pBT3-STE- VvCNGC15 and prey particles pPR3-N- VvNRT1.1 Strawberry strain NMY51 was streaked onto YPDA agar plates and incubated at 30°C for 2-3 days. Single colonies of NMY51 with a diameter of 2-3 mm, grown for 2-3 days, were picked from the YPDA plates to prepare competent yeast cells. Plasmids were co-transformed into the competent NMY51 cells. Plasmas grown to 2-3 mm on two-cell plates were added to 100 μl of 0.9% NaCl and serially diluted. The resulting bacterial solutions were then spotted onto DDO, TDO / X, and QDO / X agar plates and incubated upside down at 30°C for 3-5 days until colonies appeared.

[0055] (2) BiFC detection of the interaction between VvNRT1.1 and VvCNGC15 proteins Will VvNRT1.1 and VvCNGC15 The full-length gene is fused to the N-terminus or C-terminus of the YFP protein. The two constructed vectors are co-injected into tobacco leaves, and the fluorescence signal intensity is detected and compared with the control using a laser confocal microscope.

[0056] (3) Co-IP verification of the interaction between VvNRT1.1 and VvCNGC15 proteins Will VvNRT1.1 and VvCNGC15 Genes were extracted from tobacco leaves by Agrobacterium tumefaciens infection, followed by protein extraction via incubation (IP). Beads were then washed, and antibodies were bound to them. These antibodies then bound to bait proteins. The resulting protein groups (IP group: VvNRT1.1-4xMYC+GFP, A-4xMYC+VvCNGC15-GFP; IP group: VvNRT1.1-4xMYC+GFP, VvNRT1.1-4xMYC+VvCNGC15-GFP) were prepared by boiling and elution for Western blot analysis.

[0057] Yeast two-hybrid Y2H assays showed that VvNRT1.1 and VvCNGC15 proteins interact in vitro (see attached). Figure 3 A) BiFC verified the interaction between VvNRT1.1 and VvCNGC15 on the cell membrane, and in NO3. – Under the condition that NO3 is present, the interaction between the two weakens, indicating that NO3 – Processing can affect the interaction strength between VvNRT1.1 and VvCNGC15 (see appendix) Figure 3 B). Further verification using Co-IP shows that VvNRT1.1 and VvCNGC15 do indeed interact (see attached). Figure 3 C).

[0058] Example 5: Following the method described in Example 3, the coding sequences of VvNRT1.1 and VvCNGC15 were respectively constructed into the pBWA(V)KS-35S-osgfp vector to obtain... VvNRT1.1 and VvCNGC15 The gene overexpression vectors were named pBWA(V)kS-VvNRT1.1 and VvCNGC15, respectively. Protoblasts suitable for transformation were transferred to culture medium and cultured in the dark at 26°C for one week. Agrobacterium tumefaciens inoculum was prepared. The protoblasts were then inoculated into the prepared inoculum for 5 minutes, followed by co-culture on a co-culture medium in the dark for 3 days. The co-cultured protoblasts were then screened onto solid culture medium and cultured in the dark at 26°C until embryogenic callus appeared. The resulting embryogenic callus was transferred to somatic embryo induction medium for further screening until somatic embryos with stems appeared, and cultured in the dark at 26°C. When the shoots grew to approximately 2-3 cm, they were transferred to rooting medium for rooting. Rooting was carried out at 25°C for 16h / 8h light / dark cycles for 21 days. Positive seedlings were identified. The tested positive seedlings were hardened off for 3-4 weeks.

[0059] A total of 26 plants were obtained from the transformation. VvNRT1.1-OE and 23 strains VvCNGC15-OE Regenerated strains, obtained using molecular techniques VvNRT1.1-OE and VvCNGC15-OE The regenerated lines were tested. qRT-PCR was used (via the RT-Vv assay in Table 1). NRT1.1 and VvCNGC15 Primer, RT-Vv Actin7 Used as an internal reference gene. Expression was detected in 26 and 23 independent lines, respectively (see attached). Figure 5 Select from C and D) VvNRT1.1 and VvCNGC15 The transcriptional level was significantly higher than that of the wild-type overexpression lines, which was used for subsequent functional analysis (see appendix). Figure 6 and 7 ).

[0060] Appendix Figure 6 This indicates that under normal and low N conditions, the NO3 content of VvNRT1.1-OE and VvCNGC15-OE transgenic grape seedlings... – The absorption rates of VvNRT1.1 were all higher than those of WT, indicating that VvNRT1.1 has a higher absorption rate in NO3. – The absorption function. Meanwhile, under normal and low N conditions, the Ca of VvNRT1.1-OE and VvCNGC15-OE transgenic grape seedlings... 2+ The absorption rate was also higher than that of WT, indicating that overexpression of VvNRT1.1 or VvCNGC15 can promote the absorption of Ca by grape roots. 2+ Absorption.

[0061] Appendix Figure 7 This indicates that overexpression in grapes VvNRT1.1 VvCNGC15 enables the encoding of nitrate reductase. NIA1 Increased expression levels were accompanied by increased activity of nitrate reductase NR, indicating that NO3- – Assimilation is enhanced; using isotopes 15 NO3 – Labeling experiments found 15 NO3 – The utilization rate has increased. This proves... VvNRT1.1 Both VvCNGC15 and VvCNGC15 can improve nitrogen utilization in grapes, and both can serve as key molecular targets for improving nitrogen utilization, with potential application value.

[0062] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of nucleotides encoding nitrate transporter VvNRT1.1 and / or nucleotides encoding cyclic nucleotide-gated ion channel VvCNGC15 in improving nitrogen utilization in grapes.

2. The application according to claim 1, characterized in that, The amino acid sequence of the nitrate transporter VvNRT1.1 is shown in SEQ ID No.

2.

3. The application according to claim 1, characterized in that, The amino acid sequence of the cyclic nucleotide-gated ion channel VvCNGC15 is shown in SEQ ID No.

4.

4. The application according to claim 1, characterized in that, The nucleotide sequence encoding the nitrate transporter VvNRT1.1 is shown in SEQ ID No.

1.

5. The application according to claim 1, characterized in that, The nucleotide sequence encoding the cyclic nucleotide-gated ion channel VvCNGC15 is shown in SEQ ID No.

3.

6. A method for improving nitrogen utilization in grapes, characterized in that, Enhancing the expression of the VvNRT1.1 and / or VvCNGC15 genes or the activity of their proteins in grapes can promote the absorption and assimilation of nitrate nitrogen in grapes.

7. The method according to claim 6, characterized in that, The nucleotide sequence of VvNRT1.1 is shown in SEQ ID No. 1, and the nucleotide sequence of VvCNGC15 is shown in SEQ ID No.

3.

8. The method as described in claim 6, characterized in that, The method for enhancing the expression of VvNRT1.1 and / or VvCNGC15 genes or the activity of proteins in grapes includes the following steps: (1) Construct a recombinant expression vector containing a nucleotide sequence encoding the VvNRT1.1 protein, and / or a recombinant expression vector containing a nucleotide sequence encoding the VvCNGC15 protein; (2) Transform grape cells, tissues or organs with the recombinant expression vector constructed in step (1); (3) Screening to obtain transgenic grape plants that overexpress the VvNRT1.1 gene and / or the VvCNGC15 gene.

9. The method as described in claim 8, characterized in that, The recombinant expression vector has a backbone vector of pBWA(V)KS and a promoter of CaMV 35S.

10. The application of biomaterials containing enhanced VvNRT1.1 and / or VvCNGC15 gene expression in improving nitrogen utilization in grapes; characterized in that, The biological materials include expression cassettes, vectors, host bacteria, nucleic acid molecules, or reagent kits.

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