Application of BnIAA19-related biological product in regulating and controlling wax content and / or drought resistance of oilseed rape epidermis

Abstract

The invention belongs to the technical field of plant molecular breeding, and particularly relates to an application of a BnIAA19-related biological product in regulating and controlling the wax content and / or drought resistance of oilseed rape epidermis, and the BnIAA19 refers to any one of the following: 1) a nucleotide sequence as shown in SEQ ID NO.1; 2) a nucleotide sequence having at least 90% of identity with the SEQ ID NO.1; and 3) a nucleotide sequence capable of being hybridized with SEQ ID NO.1. The invention discloses a key negative regulation effect of BnIAA19 in regulation of epidermis wax synthesis and drought resistance, and the leaf epidermis wax content can be remarkably improved by knocking out the gene, so that the leaf water loss rate is effectively reduced, the water retention capability and survival rate of plants under drought stress are improved, and the photosynthetic water utilization efficiency is synergistically improved; the discovery provides a clear new target for stress-resistant breeding of oilseed rape and cruciferae crops, accurate regulation and control can be realized by utilizing mature technologies such as CRISPR / Cas9 and the like, and the method has the advantages of strong operability and wide application prospect.

Description

Application of BnIAA19-related bioproducts in regulating rapeseed epidermal wax content and / or drought resistance Technical Field

[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to the application of a BnIAA19-related biological product in regulating the wax content of rapeseed epidermis and / or drought resistance. Background Technology

[0002] Rapeseed (Brassica napus L.) is an important oilseed crop, playing a crucial role in edible oil supply and bioenergy development. However, its growth, development, and yield formation are highly susceptible to abiotic stresses such as drought. With the intensification of global climate change, the frequency, intensity, and extent of droughts are all increasing, severely restricting stable and high rapeseed yields. Therefore, breeding rapeseed varieties with strong drought resistance and high water use efficiency has become an important research direction in rapeseed genetics and production.

[0003] Plant epidermal wax is a hydrophobic protective structure covering the surface of plant above-ground organs, mainly composed of long-chain fatty acids and their derivatives, such as alkanes, alcohols, ketones, and aldehydes. This waxy layer effectively reflects some radiation, reduces non-stomatal water loss, prevents pathogen infection, and plays a crucial role in plant adaptation to stresses such as drought, strong light, and low temperatures. Existing research has shown that increasing the epidermal wax content of crops through genetic improvement or molecular breeding significantly enhances their water retention capacity, leaf durability, and overall survival rate under drought conditions.

[0004] Current research on the synthesis and regulation of plant epidermal waxes has made some progress, with several wax synthesis-related genes, such as CER1, KCS, and WSD1, identified and functionally validated in model crops like Arabidopsis, rice, and maize. However, in rapeseed, especially regarding the upstream signaling pathways and transcriptional regulatory networks regulating wax synthesis, little is known. Existing techniques largely focus on overexpressing or inhibiting known wax synthase genes, but these methods often have limited regulatory efficiency, are prone to causing unintended metabolic shifts, and lack a deep understanding of the global regulatory mechanisms of wax synthesis.

[0005] Therefore, there is still an urgent need to discover new key regulatory genes located upstream of wax synthesis and to establish technical solutions that can precisely regulate their expression. This will not only increase the wax content of rapeseed epidermis and enhance drought resistance, but also ensure the normal agronomic traits of the plant, providing new targets and effective technical pathways for rapeseed stress-resistant breeding. Summary of the Invention

[0006] This invention provides the application of BnIAA19-related biological products in regulating the wax content of rapeseed epidermis and / or drought resistance, solving the problem that existing genetic transformation and gene editing methods are limited in their application in rapeseed and difficult to achieve precise trait improvement.

[0007] The technical solution adopted in this invention is as follows: This invention provides the application of BnIAA19-related biological products in regulating the wax content and / or drought resistance of rapeseed epidermis, wherein BnIAA19 refers to any one of the following: 1) the nucleotide sequence shown in SEQ ID NO.1; 2) a nucleotide sequence having at least 90% identity with SEQ ID NO.1; 3) a nucleotide sequence that can hybridize with SEQ ID NO.1.

[0008] Preferably, the biological product refers to any one of the following: A) a nucleic acid molecule that alters the expression level of BnIAA19; B) a recombinant vector that alters the expression level of BnIAA19; or C) a recombinant microorganism that alters the expression level of BnIAA19.

[0009] Preferably, the nucleic acid molecule is DNA or RNA; the RNA includes any one of miRNA, siRNA, and lncRNA.

[0010] Preferably, the recombinant vector that alters the expression level of BnIAA19 refers to a recombinant vector that increases or decreases the expression level of BnIAA19; the recombinant vector that increases the expression level of BnIAA19 includes any one of pCAMBIA1300, pK2GW7, and pMDC32; the recombinant vector that decreases the expression level of BnIAA19 includes any one of CRISPR gene editing vectors and RNAi vectors.

[0011] Preferably, the starting vector for the CRISPR gene editing vector is pKSE401.

[0012] Preferably, the sequence of the sgRNA used to construct the CRISPR gene editing vector is shown in SEQ ID NO.2.

[0013] Preferably, the starting strain of the recombinant microorganism is Agrobacterium.

[0014] Preferably, the rapeseed epidermal wax content includes at least one of the following: a) alkane content; b) secondary alcohol content; c) ketone content; d) aldehyde content; e) primary alcohol content; f) fatty acid content.

[0015] Preferably, the regulation refers to any one of the following: ① increasing the expression level of BnIAA19 to reduce the wax content in the rapeseed epidermis and / or reduce the drought resistance of rapeseed; ② decreasing the expression level of BnIAA19 to increase the wax content in the rapeseed epidermis and / or improve the drought resistance of rapeseed.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention provides the application of a BnIAA19-related biological product in regulating the epidermal wax content and / or drought resistance of rapeseed. BnIAA19 refers to any one of the following: 1) the nucleotide sequence shown in SEQ ID NO.1; 2) a nucleotide sequence with at least 90% identity to SEQ ID NO.1; 3) a nucleotide sequence capable of hybridizing with SEQ ID NO.1. This invention is the first to discover the key negative regulatory role of the rapeseed BnIAA19 gene in regulating epidermal wax synthesis and drought resistance. Knocking out this gene can significantly increase the epidermal wax content of leaves and form a denser wax crystal structure, thereby effectively reducing leaf water loss rate, improving the plant's water retention capacity and survival rate under drought stress, and synergistically improving photosynthetic water use efficiency. This discovery provides a clear new target for stress resistance breeding of rapeseed and cruciferous crops, and precise regulation can be achieved using mature technologies such as CRISPR / Cas9, offering advantages of strong operability and broad application prospects. Attached Figure Description

[0017] Figure 1 shows the molecular identification results of rapeseed plants with BnIAA19 gene knockout.

[0018] Figure 2 shows the qRT-PCR results of BnIAA19 transcription level detection in BnIAA19 knockout rapeseed plants.

[0019] Figure 3 shows the qRT-PCR results of BnIAA19 transcription level detection in rapeseed plants overexpressing the BnIAA19 gene.

[0020] Figure 4 shows the comparison results of the epidermal wax content of different rapeseed materials.

[0021] Figure 5 shows the scanning electron microscope observation results of wax crystals on the leaf epidermis of different rapeseed materials.

[0022] Figure 6 shows the comparison results of leaf water loss rate of different rapeseed materials.

[0023] Figure 7 shows the characterization results of different rapeseed materials under drought stress. A: Growth status of different rapeseed varieties; B: Statistical results of relative water content of leaves of different rapeseed varieties; C: Statistical results of survival rate of different rapeseed varieties.

[0024] Figure 8 shows a comparison of transpiration rates and photosynthetic water use efficiency (SWA) of different rapeseed materials under normal water supply and drought stress conditions. A: Transpiration rate; B: Photosynthetic water use efficiency. Detailed Implementation

[0025] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0026] The inventive concept of this invention is as follows: The auxin signaling pathway is widely involved in the regulation of plant growth, development, and environmental adaptation, among which members of the Aux / IAA gene family play important regulatory roles in various plant physiological processes. However, there are no publicly available technical solutions in the prior art for increasing the epidermal wax content of rapeseed and using it for drought resistance improvement by regulating Aux / IAA genes, especially lacking research on the application of the rapeseed BnIAA19 gene in epidermal wax regulation and drought resistance breeding.

[0027] Based on this, this invention reveals and utilizes for the first time the upstream transcriptional regulatory function of BnIAA19 in the rapeseed epidermal wax synthesis pathway, providing an application of a bioproduct related to this gene in regulating rapeseed epidermal wax content and / or drought resistance. Experiments have demonstrated that downregulating or knocking out BnIAA19 can positively coordinate the expression of multiple wax synthesis-related genes, thereby significantly increasing the total amount of wax and optimizing its crystal structure. Ultimately, without affecting normal plant growth, it systematically improves rapeseed's water retention capacity, drought survival rate, and photosynthetic water use efficiency. This provides a new, efficient, precise, and easy-to-operate target and complete technical solution for molecular breeding of rapeseed stress resistance.

[0028] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0029] Example 1: Application of BnIAA19-related biological products in regulating the wax content and / or drought resistance of rapeseed epidermis, as follows: 1. Construction of BnIAA19 gene knockout rapeseed material.

[0030] A specific sgRNA target was designed based on the rapeseed BnIAA19 gene sequence. This sgRNA targets the coding region of the BnIAA19 gene and mediates CRISPR / Cas9 gene editing. The sequence of the BnIAA19 gene described in this invention is shown in SEQ ID NO.1, and the sequences of the sgRNA are shown in SEQ ID NO.2 and SEQ ID NO.3. Those skilled in the art can also select different sgRNA targets based on the BnIAA19 gene sequence using conventional design principles to achieve the same editing effect.

[0031] SEQ ID NO.1:

[0032] SEQ ID NO. 2: TGAGCTGAGACTAGGTTCTCCGG.

[0033] SEQ ID NO. 3: GGTTGCGAAAAGTCAGGTGGTGG.

[0034] sgRNA was cloned into the Cas9 vector pKSE401 to construct a CRISPR / Cas9 gene editing vector. This vector was transformed into *E. coli* DH5α competent cells using the conventional CaCl2 method. Positive clones were obtained through screening and PCR verification. The verified recombinant plasmid was transformed into *Agrobacterium* GV3101 competent cells using a freeze-thaw method, and then transformed into rapeseed hypocotyls via *Agrobacterium*-mediated transformation to obtain regenerated plants.

[0035] Through resistance screening, PCR and sequencing analysis, rapeseed plants KO#12 and KO#19 with deletion mutations in the BnIAA19 coding region were identified, as shown in Figure 1. The mutations caused frameshifts or premature termination of the BnIAA19 gene coding sequence, thereby affecting its normal protein function.

[0036] The transcription of the BnIAA19 gene in the mutant plants was further detected using qRT-PCR. The results showed that the transcription level of the BnIAA19 gene in the mutant plants was significantly different compared with that of the wild-type rapeseed plant ZS11, suggesting that mutations in the coding region may affect gene transcription stability. The results are shown in Figure 2.

[0037] The above results indicate that CRISPR / Cas9 technology can effectively obtain rapeseed plants with impaired BnIAA19 gene function, providing a material basis for subsequent trait analysis.

[0038] 2. Effects of changes in BnIAA19 expression levels on the wax content and water retention capacity of rapeseed epidermis.

[0039] To verify the function of the BnIAA19 gene, the BnIAA19 coding sequence shown in SEQ ID NO.1 was cloned into a plant expression vector to construct a BnIAA19 overexpression recombinant plasmid. The plant expression vector used in this invention is pCAMBIA1300; however, other plant expression vectors with the same function as pCAMBIA1300 can also be used, and this invention does not impose further limitations. The overexpression recombinant plasmid was introduced into Agrobacterium-competent cells GV3101 and transformed into rapeseed hypocotyls using Agrobacterium-mediated transformation to obtain regenerated plants. After resistance screening and molecular detection, multiple positive transgenic plants were obtained. qRT-PCR analysis revealed two independent overexpression lines with high BnIAA19 expression levels among the transgenic plants, named OE#5 and OE#8, for subsequent phenotypic and functional analysis. The results are shown in Figure 3.

[0040] Wild-type rapeseed plants (ZS11), BnIAA19 gene knockout rapeseed plants (KO#12 and KO#19), and BnIAA19 gene overexpressing rapeseed plants (OE#5 and OE#8) were selected and cultured to the six-leaf stage under the same growth conditions. Chloroform extraction combined with gas chromatography was used to extract and quantify the epidermal wax content of rapeseed leaves, and repeated measurements and statistical analysis were performed. The results showed that compared with wild-type rapeseed plants, the epidermal wax content of BnIAA19 gene knockout rapeseed plants was significantly increased, while the epidermal wax content of BnIAA19 gene overexpressing rapeseed plants was significantly decreased. Further analysis of the wax components revealed significant changes in the wax composition of BnIAA19 gene knockout rapeseed plants compared with wild-type plants, with significantly increased contents of alkanes, primary alcohols, and fatty acids. Correspondingly, in rapeseed plants overexpressing the BnIAA19 gene, the contents of alkanes and fatty acids in the wax were significantly reduced, and the content of primary alcohols also showed a downward trend. Furthermore, compared with the wild type, the contents of secondary alcohols, ketones, and aldehydes in the waxy components of rapeseed plants overexpressing the BnIAA19 gene were significantly reduced. These results indicate that changes in the expression level of the BnIAA19 gene can cause synergistic changes in the total amount of epidermal wax and the content of its main components in rapeseed leaves; reduced expression is conducive to the accumulation of epidermal wax, as shown in Figure 4.

[0041] Further analysis using scanning electron microscopy revealed that the waxy structure on the leaf surface of different materials was more densely distributed in rapeseed plants with the BnIAA19 gene knockout, while the number of waxy crystals on the epidermis of rapeseed plants with the BnIAA19 gene overexpression was significantly reduced and the structure was relatively sparse (Figure 5).

[0042] Meanwhile, the leaf water loss rate of different materials was measured, and the water loss rate was calculated by measuring the weight change of detached leaves at different time points under room temperature conditions. The results showed that compared with wild-type rapeseed plants, the leaf water loss rate of BnIAA19 gene knockout rapeseed plants was significantly reduced, while the leaf water loss rate of BnIAA19 gene overexpression rapeseed plants was significantly increased (see Figure 6).

[0043] The above results indicate that the decrease in the expression level of the BnIAA19 gene is conducive to the accumulation of waxy substances in the rapeseed epidermis. The resulting dense waxy structure can effectively reduce non-stomatal water loss from the leaves and improve the plant's water retention capacity.

[0044] 3. Effects of changes in BnIAA19 expression levels on drought resistance and water use characteristics of rapeseed.

[0045] Under the same growth conditions, wild-type rapeseed plants (ZS11), BnIAA19 gene knockout rapeseed plants (KO#12 and KO#19), and BnIAA19 gene overexpression rapeseed plants (OE#5 and OE#8) were selected and watering was stopped for 13 days to apply drought stress.

[0046] During the drought treatment, the relative water content of leaves from different materials was measured; after the drought treatment, the plant survival rate was statistically analyzed. Simultaneously, under normal water supply conditions and drought stress conditions, a portable photosynthesis measurement system was used to measure the transpiration rate of functional leaves from different materials, and the photosynthetic water use efficiency was calculated based on the measured parameters.

[0047] The results showed that, under drought stress, compared with wild-type rapeseed plants, rapeseed plants with BnIAA19 gene knockout exhibited higher relative leaf water content and survival rate, while rapeseed plants with BnIAA19 gene overexpression showed significantly lower relative leaf water content and survival rate (Figure 7).

[0048] Further physiological measurements showed that under normal water supply conditions, the transpiration rate of BnIAA19 gene knockout rapeseed plants was significantly reduced, while their photosynthetic water use efficiency was significantly improved. Conversely, the transpiration rate of BnIAA19 gene overexpression rapeseed plants was increased, while their photosynthetic water use efficiency was reduced. Under drought stress, the transpiration rate of all lines decreased overall compared to normal water supply conditions. Nevertheless, compared to wild-type rapeseed plants, BnIAA19 gene knockout rapeseed plants maintained a higher photosynthetic water use efficiency under drought conditions, while BnIAA19 gene overexpression rapeseed plants showed a significant decrease in photosynthetic water use efficiency (Figure 8).

[0049] The above results indicate that changes in the expression level of the BnIAA19 gene can affect the transpiration characteristics and photosynthetic water use efficiency of rapeseed plants under different water conditions, and that a decrease in its expression helps to improve the water use of plants under drought conditions.

[0050] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0051] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. The application of BnIAA19-related biological products in regulating the wax content and / or drought resistance of rapeseed epidermis, characterized in that, The BnIAA19 refers to any of the following: 1) the nucleotide sequence shown in SEQ ID NO.1; 2) a nucleotide sequence that has at least 90% identity with SEQ ID NO.1; or 3) a nucleotide sequence that can hybridize with SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The biological product refers to any one of the following: A) a nucleic acid molecule that alters the expression level of BnIAA19; B) a recombinant vector that alters the expression level of BnIAA19; C) a recombinant microorganism that alters the expression level of BnIAA19.

3. The application as described in claim 2, characterized in that, The nucleic acid molecule is DNA or RNA; the RNA includes any one of miRNA, siRNA, and lncRNA.

4. The application as described in claim 2, characterized in that, Recombinant vectors that alter BnIAA19 expression levels refer to recombinant vectors that increase or decrease BnIAA19 expression levels. Recombinant vectors that increase BnIAA19 expression levels include any one of pCAMBIA1300, pK2GW7, and pMDC32; recombinant vectors that decrease BnIAA19 expression levels include any one of CRISPR gene editing vectors and RNAi vectors.

5. The application as described in claim 4, characterized in that, The starting vector for CRISPR gene editing is pKSE401.

6. The application as described in claim 5, characterized in that, The sequence of the sgRNA used to construct the CRISPR gene editing vector is shown in SEQ ID NO.

2.

7. The application as described in claim 2, characterized in that, The starting strain of the recombinant microorganism was Agrobacterium.

8. The application as described in claim 1, characterized in that, The wax content of the rapeseed epidermis includes at least one of the following: a) alkane content; b) secondary alcohol content; c) ketone content; d) aldehyde content; e) primary alcohol content; f) fatty acid content.

9. The application as described in claim 1, characterized in that, The regulation refers to any one of the following: ① increasing the expression level of BnIAA19 to reduce the wax content in rapeseed epidermis and / or reduce rapeseed drought resistance; ② decreasing the expression level of BnIAA19 to increase the wax content in rapeseed epidermis and / or improve rapeseed drought resistance.

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