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Molecular Targets of Metabolic Gene Engineering to Increase Crop Yield
The increase in world population, the gradual reduction in the area of ​​arable land and the deterioration of the agricultural environment have made the problem of world food security increasingly serious. The excellent crop varieties cultivated by conventional breeding techniques have made significant contributions to solving the world food problem. However, in the past 20 years, the output of various crops has presented an embarrassing situation. There is no major breakthrough in the yield potential of newly-breeded varieties, and the potential for conventional breeding techniques to continue to increase grain output is limited. The birth and development of genetic engineering technology has opened up new ways for genetic improvement of crops. Especially for the genetic improvement of some quality traits, the application of genetic engineering technology is more effective. Genetically engineered pest-resistant, disease-resistant, and herbicide-resistant crops (soybeans, corn, and cotton, etc.) are now commercialized. Crop yield is a complex quantitative trait associated with many traits of the plant. Gene engineering aimed at increasing crop yields is obviously more difficult and complex than genetic improvement of individual quality traits. Nevertheless, with the development of biotechnology and the deepening of research on high-yield traits, high yield mechanisms, and related genes of crops, several technical strategies have been established that use genetic engineering technology to increase the output of food crops. For example, the development and heterotic use of male sterile lines improves the resistance (resistance) of crops to pests and environmental stresses. The development of metabolic genetic engineering allows one to modify the metabolic pathways of cells by genetically modifying the metabolism of plant cells in order to increase the accumulation of target substances or to synthesize new compounds. Photosynthesis, starch synthesis, nitrogen assimilation and water use are the basal metabolisms that form crop yields. In recent years, considerable progress has been made in the study of key steps in these metabolic pathways and genetic modification of target molecules to increase crop yields, and the understanding of plant metabolic pathways and their regulatory mechanisms has been expanded. Combining our research work, we will focus on the application of genetic engineering technology to regulate photosynthesis, starch synthesis, nitrogen assimilation, and water use and other metabolic pathways to increase crop yields. Technical strategies and research status, as well as challenges and application prospects in this research field . 1 Determination of Molecular Targets Crop yield is a complex agronomic trait, and the plant photosynthesis efficiency, nutrient absorption and utilization capacity, material transport speed, and molecular targets for defense are the first problems to be solved in the application of metabolic genetic engineering to cultivate high crop varieties. Many plant genetic studies conducted today have found that the effective regulation of plant metabolism is a complex process. In vivo metabolism of plants not only has plasticity, but also interacts with each other and is complicated. Therefore, when determining genetically modified molecular targets, several key issues often need to be considered. First, a metabolite may be a substrate for different branch metabolisms, or it may be an effector molecule that appears to be unrelated to other metabolic pathway enzymes. In this way, changing a metabolite level may have an impact on the entire metabolic network. Second, different enzymes can catalyze the same reaction, which allows plants to compensate for metabolic changes due to the genetic modification of an enzyme. In other words, even if the expression of a certain target enzyme is significantly reduced by genetic manipulation, it will not have a significant effect on metabolism. Third, plants can completely or partially silence the introduced target gene through mechanisms such as methylation and RNA interference, thus preventing or reducing the impact of the transgene on the target metabolic process. Based on the understanding of plant metabolic pathways and their regulatory mechanisms, through the analysis of the main factors affecting the distribution of metabolites (enzyme genes and their regulatory factors) and their relationship with crop yield, some genetically modified molecular targets have been established. Involved in starch synthesis, plant photosynthesis, N absorption and assimilation, water utilization and other plant physiological and metabolic processes. Many successful attempts have been made to genetically modify these molecular targets, resulting in a certain increase in crop yield, but not yet to the extent desired. At present, the genetic modification of plant metabolism with the direct purpose of increasing yield is mainly focused on the improvement of the ability of “source, flow, and bankâ€, that is, to increase the carbon source of the “bank†organization by regulating the metabolism of “source†tissues. Supply capacity; to promote the transport of photosynthetic products to the “bank†by increasing the transport capacity of assimilation products between the “source†and “libraryâ€; to increase the utilization efficiency of photocontracts by adjusting the metabolism of the “bank†organization. This in turn increases the synthesis and accumulation of specific compounds. 2 Starch-synthesized genetically modified starches are the main storage compounds in the cereal tubers and tubers of cereal crops and potato crops of cereal crops and are an important source of carbohydrates for human consumption. Every year, more than 109 tons of starches originate from rice, corn, wheat and potatoes worldwide. Enhancing the ability of starch synthesis and accumulation in tissue storage organs has always been a major goal of conventional breeding and genetic engineering breeding. Potatoes are rich in genetic resources suitable for transformation, and are easier to transform genes and obtain a large number of transgenic lines. Therefore, potato has become a model plant for studying the biochemical characteristics of metabolism related to yield and metabolic regulation. Starch is mainly synthesized in plastids. After glucose-6-phosphate (G-6-P) and glucose-1-phosphate (G-1-P) enter the plastid, under the catalysis of ADP-glucose-pyrophosphorylase (AGPase), G-1-P ATP forms ADP-glucose. ADP-glucose is a substrate for starch synthase and a precursor for starch synthesis. In early studies, the preferred strategy for genetic manipulation to increase starch synthesis was to increase ADP-glucose content. This can be achieved by genetically modifying the properties of the AGPase enzyme to increase the level of ADP glucose. This can also be achieved by altering the level of known enzymatically sufficient effectors of this enzyme, or by increasing the ADP glucose upstream metabolite, hexose phosphate. Although these genetic modification strategies greatly altered the metabolism of the transgenic plants, only the genetic modification of the AGPase enzyme properties succeeded in increasing the ADP-glucose content, which in turn increased starch synthesis and accumulation in maize kernels and potato tubers. Tjaden et al. and Loef et al. reported that the level of adenine in plastids is very important for starch synthesis. The expression of ATP/ADP transporters in potato tubers is associated with changes in starch content, and overexpression of the plastid ATP/ADP transporter leads to an increase in starch levels in tubers. However, antisense inhibition of this transporter reduced starch accumulation in tubers. It was further found that the use of adenine solution in the cultivation of potato tubers caused an increase in adenine pools in the cells, which ultimately led to an increase in starch synthesis rate in the tubers. Adenylate kinase catalyzes the conversion of ATP, AMP, and ADP, which are key enzymes that maintain various adenylate homeostasis. Therefore, adenylate kinase becomes a target molecule that regulates the size of adenine pools. Regierer et al. reduced the activity of the enzyme by modifying the plastid adenylate kinase gene, resulting in an increase in adenylate content in the transfer tuber. Transgenic potato lines had a 60% increase in starch content and a 39% increase in tuber yield compared to non-transgenic controls. Not only that, some of the amino acid content in transgenic potato tubers was also higher than in wild-type potatoes. This is one of the most successful examples of potato tuber starch content increase by genetic modification technology so far, and a new technology strategy has been established by genetically modifying the content of cofactors involved in target metabolism to increase yield. Unlike strategies commonly used to increase target metabolite yields by increasing the content of precursors of target metabolites, this strategy modifies the target as a metabolite of cofactors that not only participate in the target metabolic response, but also participate in more Other biochemical reactions. Smidansky, ED, et al. recently reported the results of the analysis of transgenic wheats that were derived from human shrunken 2 genes. The maize shrunken 2 gene encodes a large subunit of glucose pyrophosphorylase, which can reduce its sensitivity to the negative allosteric effector orthophosphate. Compared to the wild type, the average weight of wheat per plant increased by 38%. This may be due to a change in the glucose content of adenosine diphosphate, but it cannot exclude the effect of the transgene on the pleiotropic properties of other enzymes or metabolism of starch. This increase in production is thought to be due to the increase in library strength resulting from the increase in starch synthesis. It should be noted that this may be a special case of genotype-specific reactions. Because many existing tests have shown that the increase in library strength has not automatically increased potato production. As another example, the genetic modification of the mitochondrial pathway unexpectedly increased the starch content in the tubers, and the transgenic plants increased by 45% over the wild type but did not affect the total tuber yield. These studies indicate that if we can identify appropriate genetic modification targets, then there is good prospect for increasing crop yield through metabolic genetic engineering. However, due to the limitations of existing analytical tools, our interpretation of the effects of transgenes is limited. Under normal circumstances, only the specific genetic manipulation of some key metabolic components can be evaluated. For example, the cofactor library can regulate a variety of complex targets, acting on many aspects of metabolic pathways, and the explanation for the increase in tuber yield of the above-mentioned transgenic potato cannot be clear whether it is a direct or indirect result of a decrease in adenylate kinase activity. The flawed understanding of these causal mechanisms makes it difficult to assess whether genetic manipulation of the same target can produce the same effect across different crop varieties and species. 3 The genetic modification of photosynthesis to increase the photosynthetic rate of plants is a major goal of genetic modification of photosynthesis metabolic pathways. It has long been desirable to introduce C4 high photosynthetic characteristics into C3 plants to increase their photosynthetic efficiency. The key enzymes of the C4 photosynthetic pathway are phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME) and pyruvate phospho-dikinase (PPDK). The PPDK gene of C4 plant corn was transferred into potato, and the PPDK activity of transgenic plants was 5.4 times higher than that of the control. Transfection of the entire maize PEPC gene into C3 plant rice yielded transgenic rice plants that expressed corn PEPC at high levels, with PEO activity 11O times greater than the control. The C4 enzyme activity of most of the existing transgenic plants, although higher than that of the C3 control plants, is much lower than that of the C4 plants. So far there is no evidence that the C4 photosynthetic pathway has been obtained to increase production. It seems that the C4 enzyme can exert its high photosynthetic efficiency only when it is combined with the highly efficient fixed CO2 cell tissue and path unique to C4 plants. Ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) is the rate-limiting enzyme that fixes the CO2 reaction. Improvement of the activity of the enzyme, especially for CO2, can increase the photosynthetic rate of plants. Rubisco consists of a large subunit (rbcL) and a small subunit (rbcS). The large subunit is encoded by the chloroplast gene rbcL and the small subunit is encoded by the nuclear gene rbcS. Helianthus annuus Plant Rubisco has the highest CO2 affinity, about 10% higher than tobacco. The sunflower rbcL gene was introduced into tobacco chloroplasts. The transgenic tobacco chloroplasts synthesized a hexameric hybrid Rubisco composed of eight large sunflower subunits and eight small tobacco subunits. The hybrid Rubisco has an increased affinity for CO2, but not as high as sunflower Rubisco has affinity for CO2 and is easily disintegrated. Therefore, the photosynthetic efficiency of these transgenic tobacco plants did not increase. This may be due to the affinity of sunflower Rubisco large subunit and tobacco Rubisco small subunit. Rubisco of photosynthetic bacteria Phodospirillum rubrum consists of only one gene, rbcM, which is a homodimer. When rbcM was introduced into tobacco chloroplasts, Rubisco was not formed in the transgenic tobacco chloroplasts, Rubisco in R. rubrum was produced in large amounts, and it had the same CO 2 affinity as the original R. rubrum. Under conditions of CO2 enrichment, transgenic tobacco plants will grow normally. The exploration of these molecular modifications of Rubisco has accumulated experience for photosynthesis improvement. With the development of the technology, it is possible to introduce all the genes encoding the Rubisco size subunits of sunflower into other low-efficiency field crops and increase their yield. People also genetically modify other enzymes involved in photosynthesis and cofactors to increase the photosynthetic efficiency of plants. Photosynthesis CO2 fixed by the Calvin cycle quickly used to synthesize starch and other carbohydrates in order to ensure the smooth progress of photosynthesis. Accelerating the synthesis of the Calvin cycle product to the final product increases the photosynthetic rate. The reaction catalyzed by fructose-1,6-bisphosphatase is the branch point where the photosynthetic product leaves the Calvin cycle and enters the synthesis of the final product. Therefore, FBPase becomes a molecular target for gene modification. The FBPase gene isolated from cyanobacteria was introduced into tobacco so that it was expressed in chloroplasts. With 360 ppm CO2 air, the photosynthetic efficiency and growth of the transgenic plants were significantly improved. Compared with the non-transgenic control, the dry matter and CO2 fixation rate of the transgenic plants increased 1.5-fold and 1.24-fold, respectively, and Rubisco activity increased 1.2-fold. Calvin cycle intermediates and carbohydrate accumulation were all increased compared with the control. This is the first report on transgenic expression of a single plastid target enzyme to increase carbon assimilation rates and growth in recipient plants, demonstrating the feasibility and effectiveness of transgenic control of photosynthesis to increase crop yield. 4 Nitrogen uptake by genetically modified plants with nitrogen assimilation and nitrogen use efficiency have important effects on yield. Combined with the application of transgenic technology and physiological and biochemical analysis, the understanding of the molecular control mechanisms of nitrogen uptake, assimilation, and reuse during plant growth and development has become more in-depth. Glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) are major enzymes involved in nitrogen assimilation metabolism in higher plants. GS can be divided into two categories, which are mainly found in the plastids of the photosynthetic tissues and the cytoplasm of the root cells. The plastid GS is encoded by one gene and the cytoplasmic GS is encoded by a multiple gene family. Plastid GS assimilates ammonia produced by nitrate reduction and photorespiration, whereas cytosolic GS mainly assimilate ammonia produced by nitrate reduction in the plant root system. GOGAT is also divided into two groups, whose composition, molecular weight, reduction specificity and function are significantly different. GOGAT (Fd-GOGAT), which is dependent on ferredoxin, is mainly present in the cytoplasm of leaf tissue and participates in the light-induced pupa process. NADH-GOGAT is present in non-photosynthetic tissues and is used together with Gs to assimilate ammonium. GDH, which plays an important role in the reutilization of ammonium, is also classified into two types, that is, an NADH-dependent type existing in mitochondria and an NADPH-dependent type existing in chloroplasts. Mapping QTLs with maize inbred populations revealed that some QTLs controlling yield and three cytoplasmic GS genes were located in the same genomic interval, indicating that the GS-catalyzed nitrogen assimilation step has an important contribution to yield. A number of genetic manipulations have been performed on these enzymes to improve nitrogen assimilation efficiency and increase crop yields. The cytoplasmic GS was overexpressed in soybean roots, the total GS activity was increased by 10% to 30%, and the total amino acid content was significantly increased, but the plant growth and morphology did not change. Soybean cytoplasm GS was constitutively overexpressed in lotus plants, total GS activity in leaves increased by 50% to 80%, GS activity in roots did not change, transgenic plants accelerated development, and flowering advanced and prematurely declined. If the soybean cytoplasm GS is expressed in legume nodules, the plant stem and root biomass is increased by a factor of two. Overexpression of tobacco plastid GS gene in soybean leaves resulted in a 2-fold increase in GS activity in transformed chloroplasts, a 4-fold reduction in ammonium pools, and an increase in glutamate and glutamine levels. Total leaf protein and phenotype did not change. The antisense cytoplasmic GS gene was expressed in the phloem of tobacco plants. The cytoplasmic GS mRNA in the plant body was reduced by 20% to 30%, and the root and phloem ammonium content increased by 2 to 5 times, while the plastid GS and plant phenotype did not change. Tobacco overexpressing the antisense Fd-GOGAT gene reduced the Fd-GOGAT activity by 60% and caused ammonium toxicity. The cytoplasmic GS1 gene of wheat plays a key role in ammonium reuse in plants. Overexpression of GS1 in wheat leaves, GS1 activity is enhanced, nitrogen accumulates in the body, especially in grains, and the root and grain dry weight increase. Showed a good prospect of genetic improvement of wheat nitrogen assimilation metabolism. Transgenic plants that overexpress various GDH genes not only have increased growth but also have increased tolerance to stress, and further need to verify these favorable phenotypic changes under field conditions. It can be seen that further research is needed to increase crop yield by genetic modification of nitrogen assimilation metabolism. 5 Genetic modification of water use efficiency In most cases, low water use efficiency is a limiting factor in crop yields, and it has been a target of genetic improvement for many years. The ability to improve plant cell resistance to osmotic stress is a feasible way to increase the water use efficiency of plants. Many genes have been cloned for resistance (permeability) to osmotic stress, genetic modification of these genes, and improvement of plant water use efficiency have also made certain progress. For example, trehalose is a soluble, small molecule compound that accumulates in cells to increase osmotic potential and protects proteins and membranes. The trehalose-producing gene TPS1, which is catalyzed by yeast, was overexpressed in chloroplasts of tobacco plants. The trehalose content was increased by more than 20 times, the anti-osmotic stress capacity was enhanced, and the water use efficiency was also significantly increased. This was further applied. It has laid the foundation for improving the drought resistance and yield of field crops. The use of some other genes for molecular manipulations to improve plant water stress tolerance has also yielded many successful experiences. In the future, more comprehensive and more complex physiological and biochemical studies are needed to establish a corresponding technical system so that plants resistant to stress in the laboratory can continue to maintain and inherit this improved characteristic under field stress conditions. 6 Outlook and Prospects The potential for increasing crop yield through metabolic genetic engineering has not yet been fully understood and tapped. Although there are examples of transgenes increasing crop yields, it is still not possible to determine the true mechanism by which these metabolic genetic manipulations increase crop yields. Due to the limitations of analytical tools and levels, the causality of the effects of these metabolic gene modifications based on current biochemical knowledge is still limited to some speculation. An in-depth study of the link between metabolic pathways and regulatory patterns is expected to promote the widespread use of metabolic genetic engineering in the area of ​​increasing crop yields. As a new research field, the development of plant metabolomics will help to establish a more reasonable metabolic genetic engineering strategy to increase crop yield. The application of transgenic technology and metabolic control analysis techniques to the study of metabolic engineering can provide relevant information on the specific distribution, transport, and accumulation of metabolites in specific steps in a given pathway. At present, we are still faced with two problems of unknown factors in genotype-specific reactions and metabolic regulation in genetic transformation, which means that we cannot yet predict with certainty whether the metabolic improvement targets we have determined can be achieved through specific genetic manipulations. In order to solve the unpredictability of genetic engineering of plant metabolism, we need to establish more advanced and effective techniques to comprehensively analyze the effects of genetic modification. Recently, subcellular separation analysis and other micro-analysis techniques such as single cell sampling have been established. These new technologies help us establish and refine concepts related to metabolic pathways and metabolic networks, analyzing and judging which enzymes are the key enzymes for target metabolism and what are the important effects on the final yield, which in turn affect our understanding of metabolic genetic engineering targets. select. Conversion efficiency is another limiting factor affecting the application of transgenic technology to increase crop yields. The genetic transformation of some major cultivated crops is still not able to be carried out on a large scale, conventionally and rapidly. In the future, it is necessary to further improve gene vectors used for plant genetic transformation to make it easier to quickly obtain a large number of multi-gene transformants. Crop yield is a complex trait determined by multiple genes. Therefore, in order to increase crop yield, it is necessary to genetically modify multiple targets at the same time. The application of high-throughput technology can greatly accelerate the screening of target transformants (eg, transgenic plants with increased yield). Once yield-enhancing transgenic lines are obtained, it is then tested under field conditions whether these ideal target traits are stable. Frequently, under the field conditions, the reduction of the ideal target traits in the transgenic lines or even disappeared. This is a key link in the future of plant genetic improvement. In addition, transcription factors that regulate the expression of structural genes can often control multiple metabolic links. Some studies have shown that the genetic modification of transcription factors can more effectively promote the synthesis and accumulation of target metabolites, which has been successful in some secondary metabolic genetic engineering. For example, heterologous expression of transcription factors Lc and C1 involved in the synthesis of maize anthocyanins increased the content of flavonoid compounds in tomato fruits. We designed the transcription factor zinc finger protein so that it specifically binds to the soybean gene FAD2-1 target sequence. By closing the expression of the gene, we hope to increase the content of oleic acid in soybean seeds. The test results showed that compared with the control seeds, the oleic acid content of transgenic soybean seeds increased by 1 to 1.4 times, and even more unexpectedly, the seed weight increased by 1% to 4.3%. Although the increase in seed weight is not large and the specific molecular mechanism is not yet known, molecular manipulation of transcription factors can increase crop yields. Further identification of transcription factors involved in basal metabolism, through the genetic modification of a handful of pleiotropic transcription factors, genetic modification of multiple molecular targets will more effectively increase crop yield. Chloroplasts are important organelles for assimilation of plants. About half of the genes involved in photosynthesis are encoded by the chloroplast genome. The expression of chloroplast genes is crucial for plant development and cell survival. In recent years, research on chloroplast genome sequencing, gene function identification, and transformation technology has been active. Research in this area will be beneficial to improve the overall understanding of metabolic pathways and their regulatory networks for photosynthesis and help determine the targets for molecular manipulation. If the chloroplast transformation can be as simple and effective as the transformation of the nuclear genome, it will be easier to increase the crop yield through genetic manipulation of metabolic pathways. With the establishment and application of new technologies that can comprehensively analyze the effects of metabolite maps and metabolic pathways, our understanding of metabolic processes will be deepened and improved. An automated technology platform will be established and developed to enable people to more effectively and economically apply transgenic technology to improve crop target traits. We will move quickly, systematically and efficiently to carry out a new phase of crop genetic improvement by performing “piecemeal†genetic manipulations on individual targets that affect yield. Note: (1) References: Slightly omitted; those who need to contact E-mail or visit the Library of China National Rice Research Institute; (2) Source: Biotechnology Bulletin, No. 5, 2005; (3) Authors : Agricultural and Biological Engineering Center, Shanxi Agricultural University.