OsMYB86R is a favorable reporter gene
After transformation, it is better to identify the positive transformants as soon as possible. Reporter gene is a visible indicator of whether the target gene is transferred or not, also it can be widely used in identifying positive transgenic plants. To satisfy various research purposes, more and more requirements were raised to reporter genes. An ideal reporting gene should have the following characteristics: stable phenotype; easy detection; less background affect; low-cost; no damage on plants, environment and health [26–28].
Because of the possibility to meet the above requirements, some plant endogenous genes have been screened and used as reporter gene. Among this type genes, anthocyanin-related gene is one of the most widely used endogenous reporter genes in plants, such as maize, wheat, Arabidopsis, tobacco and tomato [27, 28]. The anthocyanin biosynthesis pathway involves many genes, and it is assumed that at least 16 genes are involved in anthocyanin biosynthesis in rice[29, 30]. Due to the limited carrying capacity of the vector, it is impossible to load all genes controlling anthocyanin synthesis into a same vector as the reporter genes. Scientists often use a compromise strategy to solve the problem: selecting the appropriate genetic background material, in which most anthocyanin synthesis genes are functional, as the transgenic host; using one to several anthocyanin genes as the reporter [28, 29, 31, 32].
OsC1/OsMYB86 is a homologous gene of the anthocyanin biosynthesis gene C1 in maize, and it is a functional gene for anthocyanin biosynthesis in rice. And the purple color phenotype controlled by OsC1/OsMYB86 are visible and stable, which indicates it is suitable to be used as a reporter gene. [18, 19, 21, 23, 24]. For better understanding and using this gene, OsMYB86R which was optimized from OsMYB86 was created, and in which the usual restriction sites in OsMYB86 CDS were removed. The revised OsMYB86R had normal function like OsMYB86, and could indicate the positive transformants quickly, directly, accurately and harmlessly in the early T0 generation. It is also easy to separate the positive plants from the next generations of transgenic plants out. Because the sequence of OsMYB86R is only 819 bp, which saves a lot of space for the loading of more and longer target gene. Moreover, the usual restriction sites in OsMYB86R have been removed, it also does not affect the freedom of restriction sites selection when the target gene is loaded. Therefore, OsMYB86R is a desirable reporter gene.
In addition, Zhongjiu B is a stable maintainer line of Indica rice with a shorter growth period than Zhonghua 11 (a commonly used Japonica variety in rice transgene). The transgenic efficiency of Zhongjiu B is higher than the two popular Indica rice Minghui 63 and 9311. And Zhongjiu B, transformed by only one reporter gene OsMYB86R, will display purple color in multi organs, which indicates that other anthocyanin pigment genes in Zhongjiu B are normal. Therefore, Zhongjiu B is a good Indica rice host for the transformation when OsMYB86R gene as a reporter.
RNAi promoted by candidate gene own promoter can be used to prepare gametophytic male sterility (GMS) efficiently
GMS is a type of male sterility caused by abnormal genes in pollen itself. The discovery or preparation of GMS mutants will be helpful to the GMS gene cloning, function analyzing, and mechanism understanding about GMS. It is helpful to elucidate the mechanism of pollen development. Gametophytic male sterility plays an important role in the male sterility seed production system of three-generation hybrid rice, in which the pollen lethal elements are gametophytic male sterility [33–36]. Therefore, the preparation of GMS also has key value in providing the new pollen lethal element.
The inherent phenotypic and genetic characteristics of GMS make it very difficult to find and breed its mutants. Only a few GMS genes were identified in rice, including RIP1[6]、mga36[8]、GT1[15]、OsImpβ1[5]、RUPO[14]. It is far behind the study of sporophytic male sterility, which is obstructive not only for elucidating the mechanism of pollen development, but also for developing new system of nuclear sterility production. Spontaneous mutation, induced mutation, T-DNA insertion mutation, gene editing and down-regulation expression, any one of them may result in GMS. The first three methods, due to the uncertainty of the target gene, will inevitably lead to a large number of mutants unrelated to GMS, and the efficiency of obtaining GMS is low; And gene editing, focusing its clear target gene, can ensure greatly to improve the efficiency of GMS mutation, but it is necessary to eliminate the transgenic positive plants in the next generation and detect the mutants again, and also to overcome the problems such as the continuous mutation by gene editing and the influence on subsequent analysis.
RNA interference (RNAi) is a technique which can effectively affect gene function by down-regulating target gene expression. The choice of promoter and target can affect RNAi specificity and efficiency. In this study, the strategy of RNAi driven by target gene own promoter was used, the OsPTD1 down-regulated GMS was obtained successfully. Except for the abnormal pollen fertility, there were no significant changes in other characters of the interfering plants. At the same time, the constitutive 35S promoter to drive RNAi of OsPTD1 gene was used. The results showed that the pollen fertility was not affected and no GMS materials were obtained.
The reason for the difference in specificity and efficiency of RNAi is as follows: interference driven by target gene own promoter not only ensures that the resulting double-stranded MicroRNAs degrade the target mRNA in a specific tissue (pollen) on time, moreover, the newly transferred own promoter may compete with the original promoter for the transcription factor and reduce the expression of the target gene. which together may specifically and efficiently decrease the expression of the target gene. While other promoters, such as 35S, may not be expressed in pollen and may not have the chance or ability scrambling for transcription factors. Therefore, the recommendation from this study is to use the own promoter to drive the RNAi of the candidate gene.
The combination of candidate gene RNAi and OsMYB86R makes the preparation and identification of GMS with high efficiency, accuracy and low cost
GMS mutants were obtained traditionally by natural mutation, induced mutation, T-DNA insertion mutation or gene editing. After mutagenesis, it is necessary to determine whether they were GMSs or not according to the genetic characteristics of their offspring. The identify strategy is to carry out genotype analysis by PCR or chemical resistance analysis by using screening marker genes in self crossing generation and hybridizing F1 generation (mutant as male parent). Thereafter, segregation distortion is used to determine the mutant is GMS. In other words, if the segregation ratio of self-bred offspring is 1:1 instead of 3:1, and the mutated loci is not transfer through pollen learned from F1, it is GMS [7, 10, 11]. For example, TMS1[12], OsGT1[15], and gaMS-1[13] were prepared and determined as GMS by using above methods. These current methods are cumbersome and involve the use of multi regents and equipment, which inevitably leads to problems of low-efficiency, high-cost, low-accuracy and sample damage. For gene editing strategy, scientists have to spend another generation remove the editing component for overcoming continuous editing, and screen out the mutant with no transgenic elements, which undoubtedly lengthens the identification time for at least one generation.
In this study, using the strategy combining RNAi technology and OsMYB86R reporting technology, a method for GMS preparation and identification at the same time was designed. by transferring the linked OsPTD1-RNAi element and OsMYB86R into Zhongjiu B with no purple color in any organ, the GMS genetic resources of OsPTD1 were prepared successfully. And the genetic identification of GMS was determined only three days after the seed germination, by using the purple color segregation distortion in the T1 and F1 progenies derived from T0 positive plants of RNAi.
There are three strategies to prepare GMS including random mutation (like spontaneous mutation, induced mutation and T-DNA insertion mutation and so on), gene editing (ZFNs, TALENs and CRISPR-Cas, etc.), and down regulation gene expression (such as RNAi and anti-RNA). The third strategy RNAi has been used in this study. Comparing with random mutation strategy, RNAi is much more efficient, less work and less cost for GMS preparation, because the burdensome mutant screening work is avoided in RNAi, while it is necessary in random mutation just because there is no definite target gene. Furthermore, RNAi strategy saves time compared to gene editing, because gene editing requires removing editing elements in generation T1 to avoid influence on flowing study by persistent editing. So, if gene editing strategy is used to produce GMS, the genetic population for segregation distortion analysis could only be constructed in T1 not in T0, while it would be finished in T0 when RNAi strategy is adopted. It means one generation time will be saved. At last, in identification, the first two strategies require PCR, resistance detection, or fluorescence testing to analyze inheritance behavior, which needs specialized equipment and expensive chemicals, and invasive sampling procedures. However, in the RNAi linked OsMYB86R strategy, we use anthocyanin-related genes as reporter genes to analyze their genetic characteristics, which is more direct, simple, timeless, harmless, low-cost and high-accuracy than commonly used methods based on PCR detection, resistance testing, or widely used report genes such as GUS, FPs, and Luc and so on.
In conclusion, the strategy for preparation and identification of GMS has the advantages of harmless, short-time, high-efficiency, low-cost, high-accuracy and reliability. In addition, pollen lethal factor and fluorescent protein are two key components in present technology of nuclear sterility seed production. If the GMS produced by this method aborts completely (no transgenic element transfer through pollen),the RNAi elements and linked OsMYB86R can be used to replace the pollen lethal factor and the fluorescent protein gene in the current system, which must be useful in developing new nuclear sterility seed preparation system. Obviously, this strategy can also be used for the preparation and identification of GMS in other crops. A simple sketch map for the reference to facilitate the understanding and application was shown in Fig. 4.